High intensity liquid protease formulations

Abstract

The present invention provides high intensity liquid protease compositions that are enzymatically and physically stable after storage.

Description

Technical Field

[0001] The present invention relates to enzymatically and physically stable high strength liquid protease enzyme formulations / compositions. Background Art

[0002] Industrial enzymes are used in many different industries (such as home care, food, feed, and biofuels) and are supplied as both solid and liquid products. When liquid enzyme products are shipped around the world and / or stored in warehouses, it is important that the product is stable enough to maintain specifications, even long after production when it reaches the customer. Stability includes both enzymatic stability and physical stability.

[0003] The selection of formulation ingredients for developing such stable formulations is not straightforward, as experience gained with one enzyme class is not transferable to others. Furthermore, liquid protease formulations are particularly challenging to prepare, as enzyme stability is challenged by autoproteolysis, in addition to compatibility issues with other formulation ingredients. Clearly, as the amount of enzyme protein increases, maintaining the enzyme protein in solution and maintaining a visually clear product becomes increasingly difficult. Summary of the Invention

[0004] In a first aspect, the present invention provides a liquid enzyme formulation comprising

[0005] (a) 10%-30% w / w active enzyme protein of protease,

[0006] (b) at least 40% w / w glycerol, and

[0007] (c) less than 10% w / w of formate, acetate, citrate, chloride salts or the corresponding acids; wherein the pH of the formulation is in the range of 4-7.

[0008] Other aspects and embodiments of the invention will be apparent from the description and examples.

[0009] Unless otherwise indicated, or otherwise apparent from the context, all percentages are percentages by weight (% w / w).

[0010] As used herein, the term "consisting essentially of" (and grammatical variations thereof) as applied to the compositions and methods of the present invention means that these compositions / methods may contain additional components so long as these additional components do not materially alter the composition / method.

[0011] As used herein, the term "substantially free" (and grammatical variations thereof) as applied to the compositions and methods of the present invention means that these compositions / methods may contain a small amount of a particular component, as long as the amount of the component does not materially alter the composition / method or provide any substantial effect on the composition / method. In the examples, "substantially free" means 0% w / w.

[0012] sequence

[0013] SEQ ID NO: 1: Amino acid sequence of a protease from Bacillus lentus.

[0014] SEQ ID NO: 2: Amino acid sequence of a protease from Bacillus licheniformis.

[0015] SEQ ID NO: 3: Amino acid sequence of a protease from Bacillus amyloliquefaciens.

[0016] SEQ ID NO: 4: Amino acid sequence of a protease from Bacillus gibsonii.

[0017] SEQ ID NO: 5: Amino acid sequence of a protease from Bacillus gibsonii. DETAILED DESCRIPTION

[0018] We have discovered that it is possible to prepare highly concentrated liquid protease enzyme formulations based on glycerol that are visually clear and maintain physical and enzymatic stability for weeks at room temperature.

[0019] An advantage of such concentrated liquid formulations is that the amount of solvents and other formulation ingredients is low compared to the amount of protease. This results in lower material costs, less transportation and less handling, all of which reduce the environmental impact and carbon footprint of the product.

[0020] While most other protease enzyme formulations contain fossil-based polyols such as (mono)propylene glycol, the liquid formulation of the present invention is based on glycerol, which is derived from plants and produced as a by-product of biofuel production.

[0021] It is known that both physical stability and enzyme stability are difficult under high enzyme concentrations and particularly under high protease concentrations, because protease can be self-degraded by autoprotein hydrolysis, thereby causing enzyme stability to decrease. Known (mono) propylene glycol (MPG) can stabilize subtilisin and is used as the preferred solvent for most commercial liquid protease products. As Joo et al., " Stabilization method of an alkaline protease from inactivation by heat, SDS and hydrogen peroxide [stabilization method of alkaline protease deactivated by heat, SDS and hydrogen peroxide] ", Enzyme and Microbial Technology [enzyme and microbial technology] 36 (2005), propylene glycol provides a more excellent stability than glycerol in some liquid compositions. However, contrary to expectation, the inventors found that under high protease concentrations, glycerol is a better protease solvent (see Example 2) than propylene glycol.

[0022] Physical stability is the ability of a composition to maintain a transparent, preferably clear state. This can be assessed visually or by centrifugation. For example, a liquid composition can be centrifuged at 1200 G for 10 minutes to determine whether a precipitate (solid phase) forms. Alternatively, transparency can be measured as turbidity or haze by measuring NTU using a turbidimeter to determine light scattering at 25°C (see also U.S. EPA Method 180.1).

[0023] Enzyme stability is the ability to retain enzyme activity after storage. This can be determined by measuring enzyme activity before and after storage (e.g., 4 weeks at 25°C) to determine how much activity is lost. For practical purposes, residual activity can be determined by comparing the activity of a stored sample with that of a frozen reference sample, both of which are analyzed simultaneously to eliminate day-to-day variations in the assay.

[0024] Liquid enzyme composition

[0025] The liquid enzyme formulation of the present invention comprises

[0026] (a) 10%-30% w / w active enzyme protein of protease,

[0027] (b) at least 40% w / w glycerol, and

[0028] (c) less than 10% w / w of formate, acetate, citrate, chloride salts or the corresponding acids;

[0029] The pH of the formulation is in the range of 4-7.

[0030] The liquid formulation has excellent physical stability after storage (e.g., 4 weeks at 25° C.). In preferred embodiments, the liquid formulation is visually clear; or substantially free of solid phase after centrifugation at 1200 G for 10 minutes; or has a turbidity of less than 100 NTU, such as less than 50 NTU or less than 20 NTU, as measured using a turbidimeter.

[0031] In an embodiment, the formulation is substantially free of benzoates, sorbates, sulfites, phenoxyethanol, and isothiazolinones (such as methylisothiazolinone, chloromethylisothiazolinone, benzisothiazolinone, octylisothiazolinone, dichlorooctylisothiazolinone, and butylbenzisothiazolinone).

[0032] As mentioned above, the liquid formulation also maintains excellent enzyme stability. After storage at 25°C for 4 weeks, the residual enzyme activity can be at least 90%.

[0033] Liquid enzyme formulations may contain more than 20% w / w (e.g., 20%-50% w / w) of water; preferably more than 30% w / w (e.g., 30%-50% w / w) of water, or more than 40% w / w (e.g., 40%-50% w / w) of water. The water content depends on the other ingredients of the formulation, as the total amount cannot exceed 100% (water added to 100% w / w).

[0034] Small amounts of antioxidants or reducing agents (such as sulfites, thiosulfates, nitrites, ascorbic acid / ascorbate, etc.) can also be used to stabilize the protease (and the aqueous phase in general). Other well-known stabilizers include divalent cations, such as water-soluble magnesium and calcium salts.

[0035] Protease

[0036] The protease used in the liquid formulations of the present invention is a catalytic protein, and the term "active enzyme protein" is defined herein as the amount of the catalytic protein that exhibits proteolytic activity. This can be determined using an activity-based analytical enzyme assay. In such an assay, the protease typically catalyzes a reaction that produces a colored compound. The amount of the colored compound can be measured and is related to the concentration of the active enzyme protein. This technology is well known in the art.

[0037] The protease may be a serine protease, such as subtilisin.

[0038] The protease may be a naturally occurring protease of bacterial or fungal origin, or it may be a variant derived from one or more naturally occurring proteases by gene shuffling and / or by substitution, deletion or insertion of one or more amino acids, including chemically modified mutants or protein engineered mutants.

[0039] The serine protease may for example be of the S1 family (such as trypsin) or of the S8 family (such as subtilisin). The metalloprotease may for example be a thermolysin, such as a thermolysin from the M4 family, or another metalloprotease, such as those from the M5, M7 or M8 families.

[0040] The term "subtilase" refers to a subgroup of serine proteases according to Siezen et al., Protein Eng. 4 (1991) 719-737 and Siezen et al., Protein Sci. 6 (1997) 501-523. Serine proteases are a subgroup of proteases characterized by having a serine in the active site that forms a covalent adduct with the substrate. Subtilases can be divided into six subclasses: the subtilisin family, the thermophilic protease family, the proteinase K family, the lanthionine antibiotic peptidase family, the Kexin family, and the pyrolysin family.

[0041] Although proteases suitable for detergent use can be obtained from a variety of organisms, including fungi such as Aspergillus, detergent proteases have generally been obtained from bacteria, particularly from the genus Bacillus. Examples of Bacillus species from which subtilases are derived include Bacillus lentus, Bacillus alkalophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, and Bacillus gibsonii. Particular subtilisins include subtilisinlentus, subtilisin Novo, subtilisin Carlsberg, subtilisin BPN', subtilisin 309, subtilisin 147, and subtilisin 168, as well as, for example, protease PD138 (described in WO93 / 18140). Other useful proteases are those described, for example, in WO 01 / 16285 and WO 02 / 16547.

[0042] In an embodiment of the invention, the amino acid sequence of the subtilisin has at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5, preferably at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity.

[0043] As described above, the amino acid changes can be of a minor nature, i.e., conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically 1-30 amino acids; small amino-terminal or carboxyl-terminal extensions, such as an amino-terminal methionine residue; small linker peptides of up to 20-25 residues; or small extensions that facilitate purification by altering the net charge or another function, such as a polyhistidine stretch, an antigenic epitope, or a binding module.

[0044] Essential amino acids in polypeptides can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resulting molecules are tested for protease activity to identify amino acid residues that are critical for the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. Active sites of enzymes or other biological interactions can also be determined by physical analysis of the structure, such as by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutating putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255:306-312; Smith et al., 1992, J. Mol. Biol. 224:899-904; Wlodaver et al., 1992, FEBS Lett. 309:59-64. The identity of essential amino acids can also be inferred from alignments with related polypeptides and / or from sequence homology and conserved catalytic mechanisms with related polypeptides or polypeptides / proteins within a family of polypeptides or proteins derived from a common ancestor (typically having similar three-dimensional structure, function, and significant sequence similarity). Additionally or alternatively, protein structure prediction tools can be used for protein structure modeling to identify essential amino acids and / or active sites of a polypeptide. See, e.g., Jumper et al., 2021, “Highly accurate protein structure prediction with AlphaFold,” Nature 596:583-589.

[0045] Single or multiple amino acid substitutions, deletions and / or insertions can be made and tested using known mutagenesis, recombination and / or shuffling methods, followed by relevant screening procedures, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241:53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86:2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, CRISPR gene editing, phage display (e.g., Lowman et al., 1991, Biochemistry 30:10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46:145; Ner et al., 1988, DNA 7:127).

[0046] For purposes of the present invention, use Needleman-Wunsch algorithm (Needleman-Wunsch algorithm) (Needleman and Wunsch, 1970, J.Mol.Biol. [Journal of Molecular Biology] 48:443-453) to determine the sequence identity between two amino acid sequences as the output of " longest identity ", this algorithm is as EMBOSS software package (EMBOSS:TheEuropean Molecular Biology Open Software Suite [European Molecular Biology Open Software Suite], Rice et al., 2000, Trends Genet. [genetics trend] 16:276-277) (preferred 6.6.0 version or more recent version) in the Needle program, implemented.The parameter used is gap open penalty 10, gap extension penalty 0.5 and EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.In order to make Needle program report the longest identity, must specify non-simplification (-nobrief) option in command line.The output of " longest identity " of Needle mark is calculated as follows:

[0047] (number of identical residues × 100) / (length of alignment - total number of gaps in the alignment)

[0048] Suitable commercially available proteases include those sold under the trade names Alcalase, Duralase, Durazym, Relase, Relase Ultra, Savinase, Savinase Ultra, Primase, Polarzyme, Kannase, Liquanase, Liquanase Ultra, Ovozyme, Coronase, Coronase Ultra, Blaze, Neutrase, Everlase, Esperase, Progress Uno, Progress Key and Progress Excel (Novozymes), Maxatase, Maxacal, Maxapem, Purafect, Purafect Prime, PurafectMA, Purafect Ox, Purafect OxP, Puramax, Properase, FN2, FN3, FN4, Excellase, Eraser, Opticlean, Optimase, Preferenz P200 and Preferenz P300 (DuPont / IFF), BLAP (available in the U.S. 29 of 5352604) and its variants (Henkel AG), KAP (alkalophilic Bacillus subtilisin from Kao), and those sold by Lavergy Pro (BASF).

[0049] The liquid formulations of the present invention comprise a protease (or subtilisin) in an amount of at least 10% w / w active enzyme protein, for example at least 11% w / w, at least 12% w / w, at least 13% w / w, at least 14% w / w, or at least 15% w / w active enzyme protein. The liquid formulations comprise at most 30% w / w active enzyme protein, for example at most 25% w / w active enzyme protein.

[0050] In an embodiment, the liquid formulation comprises less than 1% w / w of other enzymes (non-proteases); preferably is essentially free of other enzymes (non-proteases).

[0051] polyols

[0052] The liquid formulation comprises at least 40% w / w glycerol, such as at least 45% w / w, at least 50% w / w, at least 55% w / w or at least 60% w / w glycerol.

[0053] In an embodiment, the liquid composition comprises less than 15% w / w, preferably less than 10% w / w, less than 5% w / w, or less than 2% w / w of (mono)propylene glycol. The liquid composition may be substantially free of (mono)propylene glycol. Preferably, the (mono)propylene glycol is 1,2-propylene glycol.

[0054] In another embodiment, the liquid composition comprises less than 15% w / w, preferably less than 10% w / w, less than 5% w / w, or less than 2% w / w of polyols other than glycerol.The liquid composition may be substantially free of polyols other than glycerol.

[0055] A polyol (or polyhydric alcohol) according to the present invention is an alcohol having two or more hydroxyl groups. A polyol typically has a molecular weight of less than 500 g / mol.

[0056] Polyols include non-sugar polyols such as glycerol, ethylene glycol, diethylene glycol, triethylene glycol, propylene glycol, dipropylene glycol, tripropylene glycol, polyethylene glycol (PEG), and sugar alcohols. Polyethylene glycol can have an average molecular weight equal to or lower than about 500. Examples of sugar alcohols are sorbitol, mannitol, erythritol, galactitol, inositol, xylitol, and ribitol.

[0057] Polyols also include sugar polyols, for example, monosaccharides and disaccharides, such as glucose, fructose, galactose, sucrose, lactose, maltose, and trehalose.

[0058] Salt

[0059] Salts and corresponding acids are commonly used as buffers in liquid formulations. However, the liquid formulations of the present invention contain such a large amount of enzyme that the side chains of the amino acid residues can provide buffering capacity to the liquid formulation without the need to add a separate buffer.

[0060] Liquid formulations contain less than 10% w / w, preferably less than 8% w / w, less than 6% w / w, less than 4% w / w, less than 2% w / w, or less than 1% w / w of a salt of formate, acetate, citrate, chloride, or the corresponding acid. The amount of salt is calculated as unhydrated salt, thus excluding any complex water (water of crystallization). Preferred salts are the sodium and potassium salts of formate, acetate, citrate, and chloride.

[0061] In embodiments, the liquid formulation comprises less than 10% w / w, preferably less than 8% w / w, less than 6% w / w, less than 4% w / w, less than 2% w / w, or less than 1% w / w of any kind of salt or corresponding acid.

[0062] Protease inhibitors

[0063] As mentioned above, proteases can be stabilized using compounds that act by temporarily reducing proteolytic activity (reversible inhibitors).

[0064] Thus, the composition of the present invention may also include a protease inhibitor, which is a reversible inhibitor of protease activity. Preferably, the protease inhibitor is a (reversible) subtilisin inhibitor. In particular, the protease inhibitor may be a peptide aldehyde, boric acid, boronic acid; or a derivative of any of these. Examples of protease inhibitors are shown in, for example, WO 96 / 041859, WO 2009 / 118375, WO 2010 / 055052, and WO2013 / 004636.

[0065] In embodiments, the protease inhibitor is phenylboronic acid or a derivative thereof, such as formyl-phenyl-boronic acid (e.g., 2-FPBA, 3-FPBA, or 4-FPBA). In specific embodiments, the protease inhibitor is 4-formyl-phenyl-boronic acid (4-FPBA).

[0066] Examples of other suitable boronic acids include: thiophene-2-boronic acid, thiophene-3-boronic acid, acetamidophenylboronic acid, benzofuran-2-boronic acid, naphthalene-1-boronic acid, naphthalene-2-boronic acid, 1-thianthreneboronic acid, 4-dibenzofuranboronic acid, 5-methylthiophene-2-boronic acid, thionaphtrene boronic acid, furan-2-boronic acid, furan-3-boronic acid, 4,4-biphenyl-diboronic acid, 6-hydroxy-2-naphthalene, 4-(methylthio)phenylboronic acid, 4-(trimethyl-silyl)phenylboronic acid, 3-bromothiopheneboronic acid, 4-methylthiopheneboronic acid, 2-naphthylboronic acid, 5-bromothiopheneboronic acid, 5-chlorothiopheneboronic acid, dimethylthiopheneboronic acid, 2-bromophenylboronic acid, 3-chlorophenylboronic acid, 3-methoxy-2-thiophene, p-methyl-phenylethylboronic acid, 2-thianthreneboronic acid, dibenzothiophene Boric acid, 4-carboxyphenylboronic acid, 9-anthrylboronic acid, 3,5-dichlorophenylboronic acid, diphenylboronic anhydride, o-chlorophenylboronic acid, p-chlorophenylboronic acid, m-bromophenylboronic acid, p-bromophenylboronic acid, p-fluorophenylboronic acid, p-tolylboronic acid, o-tolylboronic acid, octylboronic acid, 1,3,5-trimethylphenylboronic acid, 3-chloro-4-fluorophenylboronic acid, 3-aminophenylboronic acid, 3,5-bis-(trifluoromethyl)phenylboronic acid, 2,4-dichlorophenylboronic acid, and 4-methoxyphenylboronic acid.

[0067] In another embodiment, the protease inhibitor is a peptide aldehyde having the formula P-B2-B1-BO-H, wherein

[0068] B0 is an amino acid selected from the group consisting of arginine (Arg), 3,4-dihydroxyphenylalanine, isoleucine (Ile), leucine (Leu), methionine (Met), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), m-tyrosine, p-tyrosine (Tyr), and valine (Val);

[0069] B1 is an amino acid selected from the group consisting of alanine (Ala), cysteine ​​(Cys), glycine (Gly), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), proline (Pro), serine (Ser), threonine (Thr), and valine (Val);

[0070] B2 is an amino acid selected from the group consisting of alanine (Ala), arginine (Arg), capryloyldipyridinium (Cpd), cysteine ​​(Cys), glycine (Gly), isoleucine (Ile), leucine (Leu), norleucine (Nle), norvaline (Nva), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), and valine (Val);

[0071] P is an N-terminal protecting group which may be selected from formyl, acetyl (Ac), benzoyl (Bz), trifluoroacetyl, methoxysuccinyl, aromatic and aliphatic urethane protecting groups such as fluorenylmethoxycarbonyl (Fmoc), methoxycarbonyl (Moc), (fluoromethoxy)carbonyl, benzyloxycarbonyl (Cbz), tert-butoxycarbonyl (Boc), and adamantyloxycarbonyl; p-methoxybenzylcarbonyl, benzyl (Bn), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), methoxyacetyl, methylaminocarbonyl, methylsulfonyl, ethylsulfonyl, benzylsulfonyl, methylphosphoramido (MeOP(OH)(═O)), and benzylphosphoramido (PhCH2OP(OH)(═O)); and

[0072] H is hydrogen.

[0073] In a preferred embodiment,

[0074] B0 is leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr), or valine (Val);

[0075] B1 is alanine (Ala), glycine (Gly), or valine (Val);

[0076] B2 is arginine (Arg), glycine (Gly), leucine (Leu), phenylalanine (Phe), or valine (Val);

[0077] P is benzyloxycarbonyl (Cbz) or methoxycarbonyl (Moc); and

[0078] H is hydrogen.

[0079] In particular embodiments, the peptide aldehyde has the formula Cbz-Gly-Ala-Tyr-H or Cbz-Val-Ala-Leu-H.

[0080] Production

[0081] The protease enzyme that is included in the liquid preparation of the present invention is produced by fermentation and subsequent recovery process conventionally.Can make fermentation liquid (fermentation liquid / broth) experience flocculation / precipitation step to provide purified protease supernatant, and this purified protease supernatant can be carried out membrane filtration to provide concentrated protease solution subsequently.Preferably, membrane filtration comprises ultrafiltration.Concentrated protease solution can be used for producing liquid preparation of the present invention subsequently in the following method, and this method comprises that concentrated protease solution is mixed with glycerol, and optionally evaporates some water under partial vacuum, increases protease concentration.Before adding glycerol, the water in the concentrated protease solution can also be evaporated.

[0082] Depending on the desired purity, the fermentation broth (from fermentation), the protease supernatant (from flocculation), or the concentrated protease solution (from membrane filtration) can be spray dried (or freeze dried) to provide a protease powder. The protease powder can then be used to produce a liquid formulation of the present invention in the following process, which comprises mixing the protease powder with water and glycerol.

[0083] Recycle

[0084] In order to flocculate the fermentation broth, a divalent salt can be added to the fermentation broth, in particular a calcium salt and / or a magnesium salt, for example calcium chloride or magnesium chloride. A preferred embodiment is a calcium salt, in particular calcium chloride.

[0085] Many aluminum compounds are known to improve flocculation, for example, Al2(SO4)3, NaAlO2, K2Al2O4, AlCl3, Al(NO3)3, Al-acetate, and Al-formates. Particularly useful polyaluminum chlorides include those having the formula Al n (OH) m Cl( 3n-m ) compounds and polyaluminium chloride and aluminium chlorohydrate having CAS number 1327-41-9.

[0086] The flocculating salt may be added to the fermentation broth in a concentration of 0.01%-10% (w / w) / kg fermentation broth (undiluted), preferably 0.5%-10% (w / w) / kg fermentation broth (undiluted), more preferably 1%-9% (w / w) / kg fermentation broth (undiluted), in particular 2%-8% (w / w) / kg fermentation broth (undiluted).

[0087] Polymers can be used for particle agglomeration. Anionic and cationic polymers are preferred. Useful cationic polymers may be polyamines, and useful anionic polymers may be polyacrylamides. Useful polymer concentrations are generally in the range of 0.5% to 20% (w / w) per kg of fermentation broth (undiluted); preferably, in the range of 1% to 10% (w / w) per kg of fermentation broth (undiluted).

[0088] Examples of useful anionic polymers are Superfloc TM A130 (Kemira). Examples of useful cationic polymers are Polycat TM (Kemira), C521 (Kemira), and C591 (Kemira).

[0089] Flocculated cell debris can be removed by methods known in the art, such as, but not limited to, filtration, eg, drum filtration, membrane filtration, filter press final filtration, cross-flow filtration, or centrifugation.

[0090] The resulting fermentation supernatant can then be further processed or refined by methods known in the art. For example, the protein can be recovered by conventional procedures including, but not limited to, further filtration (such as ultrafiltration and diafiltration), extraction, spray drying, evaporation, precipitation, or crystallization.

[0091] use

[0092] The liquid formulation of the present invention can be used to produce enzyme detergents, enzyme feeds, foods, or as a process catalyst in various industrial processes.

[0093] Thus, a liquid detergent composition can be produced by a process comprising mixing a liquid enzyme formulation of the present invention with a surfactant and a detergent builder. The final concentration of the protease in the detergent can be 0.0001% to 1% w / w active enzyme protein, preferably 0.0005% to 0.5% w / w active enzyme protein.

[0094] Surfactants can be anionic, such as linear alkylbenzene sulfonates (LAS), isomers of LAS, such as branched alkylbenzene sulfonates (BABS) and phenylalkane sulfonates; olefin sulfonates, especially α-olefin sulfonates (AOS); alkyl sulfates (AS), especially fatty alcohol sulfates (FAS), i.e. primary alcohol sulfates (PAS), such as lauryl sulfate; alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates); paraffin sulfonates (PS), including alkane-1-sulfonates and secondary alkane sulfonates (SAS); ester sulfonates, including sulfonated fatty acid glycerides and α-sulfo fatty acid methyl esters (α-SFMe or SES or MES); alkyl succinic acid or alkenyl succinic acid, such as dodecenyl / tetradecenyl succinic acid (DTSA); diesters and monoesters of sulfosuccinic acid; fatty acid derivatives of amino acids.

[0095] The surfactant may be nonionic, such as alcohol ethoxylates (AE or AEO) (e.g., AEO series such as AEO-7), alcohol propoxylates (particularly propoxylated fatty alcohols (PFA), ethoxylated alcohols and propoxylated alcohols), alkoxylated fatty acid alkyl esters (such as ethoxylated and / or propoxylated fatty acid alkyl esters (particularly ethoxymethyl ester, MEE)), alkyl polyglycosides (APG), alkoxylated amines, fatty acid monoethanolamide (FAM), fatty acid diethanolamide (FADA), ethoxylated fatty acid monoethanolamide (EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxyalkyl fatty acid amides, or N-acyl N-alkyl derivatives of glucosamine (glucamide (GA), or fatty acid glucamide (FAGA)).

[0096] The surfactant may be a glycolipid selected from the group consisting of sophorolipids, rhamnolipids, trehalolipids and mannoerythritol lipids; or a lipopeptide, such as surfactin.

[0097] The final concentration of the surfactant may be 0.5%-40% w / w, preferably 0.5%-25% w / w.

[0098] Detergent builders can be citrates, aminocarboxylates, aminopolycarboxylates and phosphonates, alkyl succinic acid or alkenyl succinic acid, 2,2',2"-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycinediacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), 1-hydroxyethane-1,1-diylbis(phosphonic acid) (HEDP), ethylenediaminetetramethylenetetra(phosphonic acid) (EDTMPA), diethylenetriaminepentamethylenepenta(phosphonic acid) (DTMPA or DTPMPA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP) , iminodisuccinic acid (IDA), N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid (SEAS), N-(2-sulfomethyl)glutamate (SMGL), N-(2-sulfoethyl)glutamate (SEGL), N-methyliminodiacetic acid (MIDA), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA), taurine-N,N-diacetic acid (TUDA) and sulfomethyl-N,N-diacetic acid (SMDA), N-(2-hydroxyethyl)ethylenediamine-N,N',N"-triacetic acid (HEDTA), diethanolglycine (DEG), aminotrimethylene tris(phosphonic acid) (ATMP) or its salt.

[0099] The final concentration of detergent builder may be 0.5% to 40% w / w, preferably 0.5% to 25% w / w.

[0100] Additional embodiments of the present invention include:

[0101] Example 1. A liquid enzyme formulation comprising

[0102] (a) 10%-30% w / w active enzyme protein of protease,

[0103] (b) at least 40% w / w glycerol, and

[0104] (c) less than 10% w / w of formate, acetate, citrate, chloride salts or the corresponding acids;

[0105] The pH of the formulation is in the range of 4-7.

[0106] Embodiment 2. The enzyme formulation of the preceding embodiment, wherein the protease is a serine protease.

[0107] Embodiment 3. The enzyme formulation of the preceding embodiment, wherein the protease is subtilisin.

[0108] Embodiment 4. The enzyme formulation according to any one of the preceding embodiments, comprising less than 15% w / w of (mono)propylene glycol, preferably the (mono)propylene glycol is 1,2-propylene glycol.

[0109] Embodiment 5. The enzyme formulation according to any one of the preceding embodiments, comprising less than 10% w / w of (mono)propylene glycol, preferably the (mono)propylene glycol is 1,2-propylene glycol.

[0110] Embodiment 6. The enzyme formulation according to any one of the preceding embodiments, comprising less than 5% w / w of (mono)propylene glycol, preferably the (mono)propylene glycol is 1,2-propylene glycol.

[0111] Embodiment 7. The enzyme formulation according to any one of the preceding embodiments, comprising less than 2% w / w of (mono)propylene glycol, preferably the (mono)propylene glycol is 1,2-propylene glycol.

[0112] Embodiment 8. The enzyme formulation of any of the preceding embodiments, which is substantially free of (mono)propylene glycol, preferably the (mono)propylene glycol is 1,2-propylene glycol.

[0113] Embodiment 9. The enzyme formulation of any one of the preceding embodiments, comprising less than 15% w / w of polyols other than glycerol.

[0114] Embodiment 10. The enzyme formulation of any one of the preceding embodiments, comprising less than 10% w / w of polyols other than glycerol.

[0115] Embodiment 11. The enzyme formulation of any one of the preceding embodiments, comprising less than 5% w / w of polyols other than glycerol.

[0116] Embodiment 12. The enzyme formulation of any one of the preceding embodiments, comprising less than 2% w / w of polyols other than glycerol.

[0117] Embodiment 13. The enzyme formulation of any of the preceding embodiments, which is substantially free of polyols other than glycerol.

[0118] Embodiment 14. The enzyme formulation of any preceding embodiment, comprising at least 45% w / w glycerol.

[0119] Embodiment 15. The enzyme formulation of any preceding embodiment, comprising at least 50% w / w glycerol.

[0120] Embodiment 16. The enzyme formulation of any preceding embodiment, comprising at least 55% w / w glycerol.

[0121] Embodiment 17. The enzyme formulation of any preceding embodiment, comprising at least 60% w / w glycerol.

[0122] Embodiment 18. The enzyme formulation of any preceding embodiment, comprising less than 5% w / w of a salt of formate, acetate, citrate, chloride, or the corresponding acid.

[0123] Embodiment 19. The enzyme formulation of any preceding embodiment, comprising less than 2% w / w of a salt of formate, acetate, citrate, chloride, or the corresponding acid.

[0124] Embodiment 20. The enzyme formulation of any preceding embodiment, comprising less than 10% w / w salt or the corresponding acid.

[0125] Embodiment 21. The enzyme formulation of any preceding embodiment, comprising less than 5% w / w of salt or the corresponding acid.

[0126] Embodiment 22. The enzyme formulation of any preceding embodiment, comprising less than 2% w / w of salt or the corresponding acid.

[0127] Embodiment 23. The enzyme formulation of any preceding embodiment, further comprising a protease inhibitor.

[0128] Embodiment 24. The enzyme formulation of any preceding embodiment, further comprising a boronic acid protease inhibitor or a derivative thereof.

[0129] Embodiment 25. The enzyme formulation of any preceding embodiment, further comprising a phenylboronic acid protease inhibitor or a derivative thereof.

[0130] Embodiment 26. The enzyme formulation of any preceding embodiment, further comprising a formylphenylboronic acid protease inhibitor, such as 4-FPBA.

[0131] Embodiment 27. The enzyme formulation of any one of embodiments 1-23, further comprising a peptide aldehyde protease inhibitor.

[0132] Embodiment 28. The enzyme formulation of the preceding embodiment, wherein the peptide aldehyde has the formula P-B2-B1-B0-H, wherein

[0133] B0 is an amino acid selected from the group consisting of leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr) or valine (Val);

[0134] B1 is an amino acid selected from the group consisting of alanine (Ala), glycine (Gly) or valine (Val);

[0135] B2 is an amino acid selected from the group consisting of arginine (Arg), glycine (Gly), leucine (Leu), phenylalanine (Phe) or valine (Val);

[0136] P is an N-terminal protecting group, preferably benzyloxycarbonyl or methoxycarbonyl; and

[0137] H is hydrogen.

[0138] Embodiment 29. The enzyme formulation of the preceding embodiment, wherein the peptide aldehyde has the formula Cbz-Gly-Ala-Tyr-H or Cbz-Val-Ala-Leu-H.

[0139] Embodiment 30. The enzyme formulation of any preceding embodiment, wherein the protease has at least 80% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0140] Embodiment 31. The enzyme formulation of any preceding embodiment, wherein the protease has at least 85% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0141] Embodiment 32. The enzyme formulation of any preceding embodiment, wherein the protease has at least 90% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0142] Embodiment 33. The enzyme formulation of any preceding embodiment, wherein the protease has at least 95% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0143] Embodiment 34. The enzyme formulation of any preceding embodiment, wherein the protease has at least 96% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0144] Embodiment 35. The enzyme formulation of any preceding embodiment, wherein the protease has at least 97% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0145] Embodiment 36. The enzyme formulation of any preceding embodiment, wherein the protease has at least 98% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0146] Embodiment 37. The enzyme formulation of any preceding embodiment, wherein the protease has at least 99% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 5.

[0147] Embodiment 38. The enzyme formulation of any one of embodiments 30-37, wherein the amino acid change is a substitution.

[0148] Embodiment 39. The enzyme formulation of any preceding embodiment, which is substantially free of other enzymes (non-proteases).

[0149] Embodiment 40. The enzyme formulation of any preceding embodiment, which is free of preservatives.

[0150] Embodiment 41. The enzyme formulation of any preceding embodiment, which is free of benzoate, sorbate, sulfite, phenoxyethanol, and isothiazolinone.

[0151] Embodiment 42. The enzyme formulation of any preceding embodiment, wherein the composition is substantially free of solid phase after centrifugation at 1200 G for 10 minutes.

[0152] Embodiment 43. The enzyme formulation of any preceding embodiment, having a turbidity of less than 100 NTU.

[0153] Embodiment 44. The enzyme formulation of any preceding embodiment, having a turbidity of less than 50 NTU.

[0154] Embodiment 45. The enzyme formulation of any preceding embodiment, having a turbidity of less than 20 NTU.

[0155] Embodiment 46. A method for preparing the liquid enzyme formulation as described in any one of the preceding embodiments, the method comprising

[0156] (a) providing a fermentation broth comprising a protease in an amount of at least 1% w / w;

[0157] (b) subjecting the fermentation broth to flocculation and membrane filtration to provide a concentrated protease solution; and

[0158] (c) mixing the protease solution with glycerol.

[0159] Embodiment 47. The method of the preceding embodiment, further comprising:

[0160] (d) Evaporation of water under partial vacuum to increase protease concentration.

[0161] Embodiment 48. The method of embodiment 46 or 47, wherein the membrane filtration comprises ultrafiltration.

[0162] Embodiment 49. A method for preparing the liquid enzyme formulation as described in any one of the preceding embodiments, the method comprising

[0163] (a) spray drying or freeze drying a protease solution to provide a powder comprising the protease; and

[0164] (b) Mix the powder with water and glycerin.

[0165] Embodiment 50. A process for preparing a liquid detergent composition, the process comprising mixing the liquid enzyme formulation of any one of the preceding embodiments with a surfactant and a detergent builder; preferably, the detergent builder is a non-phosphorus builder / chelant.

[0166] Embodiment 51. The method of the preceding embodiment, wherein the final concentration of the surfactant is 0.5%-40% w / w, preferably 0.5%-25% w / w.

[0167] Embodiment 52. The method of the preceding embodiment, wherein the final concentration of the detergent builder is 0.5%-40% w / w, preferably 0.5%-25% w / w.

[0168] Embodiment 53. The method of the preceding embodiment, wherein the final concentration of the protease is 0.0001%-1% w / w active enzyme protein, preferably 0.0005%-0.5% w / w active enzyme protein.

[0169] Examples

[0170] Chemicals were commercial products of at least reagent grade.

[0171] Example 1

[0172] High-strength liquid protease formulation with glycerin

[0173] The following formulations (AD) were all prepared by mixing a flocculated and concentrated (by ultrafiltration) liquid protease solution with glycerol and then evaporating to >10.4% active enzyme protein (AEP) with or without the subsequent addition of water, glycerol and / or disubstituted alaninamide. The pH of the composition was adjusted as needed using acetic acid or sodium hydroxide.

[0174] Composition A

[0175] 10.4% w / w active enzyme protein (Liquanase from Novozymes)

[0176] 60% w / w glycerin

[0177] 0.325% w / w disubstituted alaninamide (peptide aldehyde protease inhibitor)

[0178] pH 5.5

[0179] Composition B

[0180] 11.6% w / w active enzyme protein (Liquanase from Novozymes)

[0181] 50% w / w glycerin

[0182] 0.36% w / w disubstituted alaninamide (peptide aldehyde protease inhibitor)

[0183] pH 5.5

[0184] Composition C

[0185] 12.3% w / w active enzyme protein (Coronase from Novozymes)

[0186] 50% w / w glycerin

[0187] pH 5.5

[0188] Composition D

[0189] 19.1% w / w active enzyme protein (Coronase from Novozymes)

[0190] 47.3% w / w glycerin

[0191] pH 5.5

[0192] result

[0193] Enzyme stability was measured as residual activity relative to a reference stored at -18°C.

[0194] Physical stability was assessed by visual inspection. "Clear" corresponds to NTU < 100.

[0195] Table 1. Stability of compositions AD.

[0196]

[0197] As shown in Table 1, compositions AD exhibited excellent stability after storage at 25°C for at least 13 weeks.

[0198] Example 2

[0199] High-strength liquid protease formulation containing propylene glycol

[0200] The following compositions (E and F) were prepared using the same procedure as in Example 1. The compositions were stored at 40°C for 2 weeks and 4 weeks prior to evaluation.

[0201] Composition E

[0202] 12% w / w active enzyme protein (Liquanase from Novozymes)

[0203] 60% w / w glycerin

[0204] 0.38% w / w disubstituted alaninamide (peptide aldehyde protease inhibitor)

[0205] pH 5.5

[0206] Composition F

[0207] 12% w / w active enzyme protein (Liquanase from Novozymes)

[0208] 40% w / w glycerin

[0209] 20% w / w propylene glycol (1,2-propanediol)

[0210] 0.38% w / w disubstituted alaninamide (peptide aldehyde protease inhibitor)

[0211] pH 5.5

[0212] Table 2. Visual assessment of the physical stability of compositions E and F.

[0213]

[0214] 'Clear' corresponds to NTU < 100 with no visible precipitate.

[0215] 'Precipitate' indicates a clearly visible precipitate.

[0216] The data in Table 2 show that Composition E, which contains only glycerin, has much better physical stability than Composition F, which contains a large amount (about 33%) of propylene glycol.

[0217] Compositions E and F were also prepared at 10.1% w / w, 10.7% w / w and 11.4% w / w active enzyme protein, and the physical stability data were consistent with that presented in Table 2.

Claims

1. A liquid enzyme formulation comprising (a) 10%-30% w / w active enzyme protein of protease, (b) at least 40% w / w glycerol, and (c) less than 10% w / w of formate, acetate, citrate, chloride salts or the corresponding acids; The pH of the formulation is in the range of 4-7.

2. The enzyme formulation according to the preceding claim, wherein the protease is a serine protease; preferably a subtilisin.

3. The enzyme formulation according to any one of the preceding claims, comprising less than 10% w / w monopropylene glycol; preferably less than 5% w / w or less than 2% w / w monopropylene glycol.

4. The enzyme formulation of any one of the preceding claims, comprising at least 45% w / w glycerol; preferably at least 50% w / w, at least 55% w / w or at least 60% w / w glycerol.

5. The enzyme formulation of any one of the preceding claims, comprising less than 10% w / w or less than 5% w / w of salt or the corresponding acid.

6. The enzyme formulation according to any one of the preceding claims, further comprising a protease inhibitor, preferably a boronic acid or a peptide aldehyde.

7. The enzyme formulation according to the preceding claim, wherein the boronic acid is phenyl-boronic acid, such as 4-formyl-phenyl-boronic acid.

8. The enzyme formulation of claim 6, wherein the peptide aldehyde has the formula P-B2-B1-BO-H, wherein B0 is an amino acid selected from the group consisting of leucine (Leu), methionine (Met), phenylalanine (Phe), tyrosine (Tyr) or valine (Val); B1 is an amino acid selected from the group consisting of alanine (Ala), glycine (Gly) or valine (Val); B2 is an amino acid selected from the group consisting of arginine (Arg), glycine (Gly), leucine (Leu), phenylalanine (Phe) or valine (Val); P is an N-terminal protecting group, preferably benzyloxycarbonyl or methoxycarbonyl; and H is hydrogen.

9. The enzyme formulation of claim 6, wherein the peptide aldehyde has the formula Cbz-Gly-Ala-Tyr-H or Cbz-Val-Ala-Leu-H.

10. The enzyme formulation of any one of the preceding claims, wherein the protease has at least 90% amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO:

5.

11. The enzyme formulation of any preceding claim, comprising less than 1% w / w of other enzymes (non-proteases); preferably being substantially free of other enzymes.

12. The enzyme formulation according to any one of the preceding claims, which is free of preservatives; preferably free of benzoates, sorbates, sulfites, phenoxyethanol and isothiazolinones.

13. The enzyme formulation of any one of the preceding claims, wherein there is substantially no solid phase after centrifuging the composition at 1200 G for 10 minutes, or the enzyme formulation has a turbidity of less than 100 NTU, preferably less than 50 NTU or less than 20 NTU.

14. A method for preparing a liquid enzyme formulation as claimed in any one of the preceding claims, the method comprising (a) providing a fermentation broth comprising a protease in an amount of at least 1% w / w; (b) subjecting the fermentation broth to flocculation and membrane filtration to provide a concentrated protease solution; (c) mixing the protease solution with glycerol; and (d) optionally evaporating water under partial vacuum to increase the protease concentration; Preferably, the membrane filtration in step (b) comprises ultrafiltration.

15. A method for preparing a liquid enzyme formulation as claimed in any one of the preceding claims, the method comprising (a) spray drying a protease solution to provide a powder comprising the protease; and (b) Mix the powder with water and glycerin.

16. A process for the preparation of a liquid detergent composition, the process comprising admixing a liquid enzyme formulation as claimed in any one of the preceding claims with a surfactant and a detergent builder.

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