STABILIZED SUBTILYSINE COMPOSITION, METHOD OF PREPARING THE COMPOSITION, AND COMPOUND FOR USE IN THE COMPOSITION

Abstract

 

Description

STABILIZED SUBTILYSINE COMPOSITION, METHOD OF PREPARING THE COMPOSITION, AND COMPOUND FOR USE IN THE COMPOSITION Reference to a Sequence Listing

[0001] This application contains a List of Sequences in a computer-readable form. TECHNICAL FIELD

[0002] The present invention relates to a composition comprising a subtilisin stabilized by a peptide aldehyde derivative. It also relates to a method of preparing the composition and to a compound for use in the composition. BACKGROUND OF THE INVENTION

[0003] WO 98 / 13458, WO 94 / 04651, WO 98 / 13460, WO 95 / 25791, and WO 2009 / 118375 disclose liquid detergents with a subtilisin-like protease stabilized by a peptide aldehyde. WO 2011 / 036153 discloses that the addition of a peptide aldehyde to a particulate detergent containing subtilisin can improve detergency.

[0004] It is known that aldehydes can form soluble adducts with NaHSO3 (bisulfite or hydrosulfite adducts) and that peptide aldehydes tend to be moderately water-soluble.

[0005] WO 98 / 47523 and US 6,500,802 disclose peptidyl-2-amino-1-hydroxyalkanesulonic acids and their use as protease inhibitors. US 5,436,229 discloses bisulfite adducts of L-arginine aldehyde derivatives and their use as thrombin inhibitors.

[0006] US 4,703,036, US 4,478,745 and US 5,578,574 disclose methods for preparing peptide aldehydes in dry form. SUMMARY OF THE INVENTION

[0007] In the large-scale production of stabilized subtilisin compositions, it is strongly preferable to handle liquid raw materials, because Petition 870190104795, dated 10 / 17 / 2019, page 6 / 63 / 29 safety and procedural reasons. Peptide aldehydes tend to be moderately water-soluble, which makes the preparation of concentrated aqueous solutions difficult or impossible and requires the use of dry product or highly dilute aqueous solutions when making stabilized subtilisin solutions for use in liquid formulations or for granulation to make granular compositions.

[0008] The conversion of peptide aldehyde into a hydrosulfite adduct can be used to increase aqueous solubility in the purification of peptide aldehydes. Advantageously, we found that this hydrosulfite adduct is effective as an inhibitor and stabilizer of subtilisin and that it can also stabilize a second enzyme (non-subtilisin), if present. We found that the hydrosulfite adduct is effective as a subtilisin inhibitor, and that it maintains its inhibitory and stabilizing effect in a liquid detergent during storage. Thus, the use of the hydrosulfite adduct can avoid the cost and time of reconverting it to peptide aldehyde and can save the subsequent drying of the peptide aldehyde, and this can avoid the inconvenience of handling peptide aldehyde in powder form or as a highly dilute aqueous solution. Furthermore, the addition of a peptide aldehyde bisulfite adduct can also improve the detergency (washing performance) of a detergent containing subtilisin.

[0009] Accordingly, the invention provides a composition comprising a subtilisin and a peptide aldehyde hydrosulfite adduct with the formula X-B1-NH-CHR-CHOH-SO3M. The groups in the formula have the following meaning:

[00010] a) M is H (hydrogen) or an alkali metal, preferably Na or K;

[00011] b) R is a group in which NH-CHR-CO is an L or D amino acid residue (hereinafter referred to as B0);

[00012] c) B1 is an amino acid residue; and Petition 870190104795, dated 10 / 17 / 2019, p. 7 / 63 / 29

[00013] d) X consists of one or more amino acid residues (preferably one or two), optionally comprising an N-terminal protecting group.

[00014] The invention further provides a method for preparing the composition, which comprises mixing a subtilisin, an aqueous solution comprising the peptide aldehyde hydrosulfite adduct with the formula X-B1-NH-CHR-CHOH-SO3M (wherein M, R, B1 and X are as defined above), and optionally a surfactant.

[00015] Additionally, the invention provides a compound for use in the composition, which is a hydrosulfite aldehyde peptide adduct with the formula X-B1-NH-CHR-CHOH-SO3M. R can be p-hydroxybenzyl, and M, B1 and X are as defined above. DETAILED DESCRIPTION OF THE INVENTION Stabilized subtilisin composition

[00016] The composition of the invention comprises a subtilisin and a hydrosulfite aldehyde peptide adduct and may optionally comprise a second enzyme. The composition may be in liquid or granular form. It may be a detergent composition further comprising a surfactant.

[00017] In a composition such as a liquid or granular detergent, the amount of each enzyme (subtilisin and optional second enzyme) will typically be 0.04-80 micro-M (or micro-mol / kg), in particular 0.2-30 micro-M, especially 0.4-20 micro-M (generally 1-2000 mg / l or mg / kg, in particular 5-750 mg / l, especially 10-500 mg / l) calculated as pure enzyme protein. In a composition such as an enzyme concentrate, the amount of each enzyme will typically be 0.01-20 mM, in particular 0.04-10 mM, especially 0.1-5 mM (generally 0.3-500 g / l, in particular 1-300 g / l, especially 3-150 g / l) calculated as pure enzyme protein. Petition 870190104795, dated 10 / 17 / 2019, page 8 / 63 / 29

[00018] The molar ratio of the enzyme stabilizer or inhibitor according to the invention with respect to subtilisin is at least 1:1 or 1.5:1, and is less than 1000:1, more preferably less than 500:1, even more preferably between 100:1 and 2:1 or between 20:1 and 2:1, or even more preferably the molar ratio is between 10:1 and 3:1. Peptide aldehyde

[00019] The bisulfite adduct used in the method can be derived from a peptide aldehyde with the formula X-B1-B0-H wherein the groups are defined as above with B0 being a single amino acid residue with L or D configuration with the formula: NH-CHR-CO.

[00020] NH-CHR-CO (B0) is an L or D amino acid residue, where R can be an aliphatic or aromatic side chain, for example, aralkyl, such as benzyl, where R can be optionally substituted. More particularly, the B0 residue can be bulky, neutral, polar, hydrophobic and / or aromatic, optionally substituted. Examples are the D or L form of Tyr (p-tyrosine), m-tyrosine, 3,4-dihydroxyphenylalanine, Phe, Val, Met, norvaline (Nva), Leu, Ile or norleucine (Nle), Tyr, m-tyrosine, 3,4-dihydroxyphenylalanine, Phe, Val, Met, Nva or Nle.

[00021] In the formula above, X-B1-B0-H, the B1 residue can be particularly small, aliphatic, hydrophobic and / or neutral. Examples are alanine (Ala), cysteine ​​(Cys), glycine (GIy), proline (Pro), serine (Ser), threonine (Thr), valine (VaI), norvaline (Nva) and norleucine (Nle), particularly Ala, Cys, GIy, Ser, Thr, VaI, Nva and Nle.

[00022] X can in particular be one or two amino acid residues with an optional N-terminal protecting group (i.e., the compound is a tri- or tetrapeptide aldehyde with or without a protecting group). Thus, X can be B2, B3-B2, Z-B2, Z-B3-B2 where B3 and B2 each represent an amino acid residue and Z is an N-terminal protecting group.

[00023] Residue B2 may in particular be small, aliphatic and / or Petition 870190104795, dated 10 / 17 / 2019, p. 9 / 63 / 29 neutral, for example Ala, Gly, Thr, Arg, Leu, Phe or Val; particularly Gly, Thr or Val.

[00024] Residue B3 may in particular be bulky, hydrophobic, neutral and / or aromatic, optionally substituted, for example, Phe, Tyr, Trp, phenylglycine, Leu, Val, Nva, Nle or Ile.

[00025] The Z protecting group at the N-terminus (if present) may be selected from formyl, acetyl (Ac), benzoyl, trifluoroacetyl, fluoromethoxycarbonyl, methoxysuccinyl, aromatic and aliphatic urethane protecting groups, benzyloxycarbonyl (Cbz), t-butyloxycarbonyl, adamantiloxycarbonyl, p-methoxybenzyl carbonyl (MOZ), benzyl (Bn), p-methoxybenzyl (PMB) or p-methoxyphenyl (PMP), methoxycarbonyl (Moc); methoxyacetyl (Mac); methyl carbamate or a methylamino carbonyl / methyl urea group. In the case of a tetrapeptide aldehyde with a protecting group (i.e., X = Z-B3-B2), Z is preferably a small aliphatic group, for example formyl, acetyl, fluoromethoxycarbonyl, t-butyloxycarbonyl, methoxycarbonyl (Moc); methoxyacetyl (Mac); methyl carbamate or a urea methylamino carbonyl / methyl group.In the case of a tripeptide aldehyde with a protecting group (i.e., X = Z- B2), Z is preferentially a bulky aromatic group such as benzoyl, benzyloxycarbonyl, p-methoxybenzyl carbonyl (MOZ), benzyl (Bn), pmethoxybenzyl (PMB) or p-metoxyphenyl (PMP).

[00026] Suitable peptide aldehydes are described in WO 94 / 04651, WO 95 / 25791, WO 98 / 13458, WO 98 / 13459, WO 98 / 13460, WO 98 / 13461, WO 98 / 13461, WO 98 / 63 98 / 13462, WO 2007 / 141736, WO 2007 / 145963, WO 2009 / 118375, WO 2010 / 055052 and WO 2011 / 036153. More particularly, the peptide aldehyde can be Z-RAY-H, Ac-GAY-H, Z-GAY-H, Z-GAL-H, ZVAL-H, Z-GAF-H, Z-GAV-H, Z-GGY-H, Z-GGF-H, Z-RVY-H, Z-LVY-H, Ac-LVY-H, Ac-GAY-H, Ac-GY-H Ac-YGAY-H, Ac-FGAL-H, Ac-FGAF-H, AcFGVY-H, Ac-FGAM-H, Ac-WLVY-H, MeO-CO-VAL-H, MeNCO-VAL-H, Petition 870190104795, of 17 / 10 / 2019, p. 10 / 63 / 29 MeO-CO-FGAL-H, MeO-CO-FGAF-H, MeSO2-FGAL-H, MeSO2-VAL-H, PhCH2O(OH)(O)P-VAL-H, EtSO2-FGAL-H, PhCH2SO2-VAL-H, PhCH2O(OH)(O)P-LAL-H, PhCH2O(OH)(O)P-FAL-H, or MeO(OH)(O)PLGAL-H. Here, Z is benzyloxycarbonyl, Me is methyl, Et is ethyl, Ac is acetyl, H is hydrogen, and the other letters represent amino acid residues denoted by standard single-letter notation (e.g., F = Phe, Y = Tyr, L = Leu).

[00027] Alternatively, the peptide aldehyde may have the formula described in WO 2010 / 055052:

[00028] PO-(Ai-X')n-An+1-Q

[00029] where Q is hydrogen, CH3, CX3, CHX2, or CH2X, where X is a halogen atom;

[00030] where one X' is the “double encapsulation group N” CO, COCO, CS, CS-CS or CS-CO, most preferably urido (CO), and the other X' are nothing,

[00031] where n = 1-10, preferably 2-5, more preferably 2,

[00032] where each of Ai and An+1 is an amino acid residue with the structure:

[00033] -NH-CR-CO- for a residue to the right of X= -CO-, or

[00034] -CO-CR-NH- for a residue to the left of X= -CO

[00035] where R is H or an optionally substituted alkylaryl or alkyl group that may optionally include a heteroatom and may optionally be bonded to the N atom, and

[00036] where P is hydrogen or any C-terminal protecting group.

[00037] Examples of such peptide aldehydes include α-MAPI, βMAPI, F-urea-RVY-H, F-urea-GGY-H, F-urea-GAF-H, F-urea-GAY-H, F-urea-GAL-H, F-urea-GA-Nva-H, F-urea-GA-Nle-H, Y-urea-RVY-H, Yurea-GAY-H, F-CS-RVF-H, F-CS-RVY-H, F-CS-GAY-H, Antipain, Petition 870190104795, dated 10 / 17 / 2019, p. 11 / 63 / 29 GE20372A, GE20372B, Chymostatin A, Chymostatin B, and Chymostatin C.

[00038] Other examples of peptide aldehydes are disclosed in WO 2010 / 055052 and WO 2009 / 118375, WO 94 / 04651, WO 98 / 13459, WO 98 / 13461, WO 98 / 13462, WO 2007 / 145963, (P&G) incorporated herein by reference. Hydrosulfite aldehyde peptide adduct

[00039] The peptide aldehyde hydrosulfite adduct can be derived from the peptide aldehydes described above.

[00040] Particular examples are Cbz-RA- NHCH(CH2C6H4OH)C(OH)(SO3M)-H, Ac-GA- NHCH(CH2C6H4OH)C(OH)(SO3M)-H, Cbz-GA- NHCH(CH2C6H4OH)C(OH)(SO3M)-H, Cbz-GA- NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, Cbz-VA- NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, Cbz-GA- NHCH(CH2Ph)C(OH)(SO3M)-H, Cbz-GA-NHCH(CH(CH3)2)C(OH)(SO3M)- H, Cbz-GG-NHCH(CH2C6H4OH)C(OH)(SO3M)-H, Cbz-GG- NHCH(CH2Ph)C(OH)(SO3M)-H, Cbz-RV- NHCH(CH2C6H4OH)C(OH)(SO3M)-H, Cbz-LV- NHCH(CH2C6H4OH)C(OH)(SO3M)-H, Ac-LGA- NHCH(CH2C6H4OH)C(OH)(SO3M)-H, Ac-FGA- NHCH(CH2C6H4OH)C(OH)(SO3M)-H, Ac-YGA- NHCH(CH2C6H4OH)(SO3M)-H Ac-FGA- NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, Ac-FGA- NHCH(CH2Ph)C(OH)(SO3M)-H, Ac-FGV- NHCH(CH2C6H4OH)C(OH)(SO3M)-H, Ac-FGA- NHCH(CH2CH(CH3)2)(SO3M)-CH Ac-WLV- NHCH(CH2C6H4OH)C(OH)(SO3M)-H, MeO-CO-VA- NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, MeNCO-VA- NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, MeGA-CO-F Petition 870190104795, of 17 / 10 / 2019, p. 12 / 63 / 29 NHCH(CH2CH(CH3)2))C(OH)(SÜ3M)-H, NHCH(CH2Ph)C(OH)(SÜ3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SÜ3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, MeO-CO-FGAMeSO2-FGAMeSO2-VAPhCH2O(OH)(O)P-VAEtSO2-FGAPhCH2SO2-VAPhCH2O(OH)(O)P-LAPhCH2O(OH)(O)P-FAMeO(OH)(O)P-LGAe F-urea-RVNHCH(CH2CeH4OH)C(OH)(SO3M)-H where M=negative charge, H, Na, or K or other contraction. Subtilisin

[00041] Subtilisins are a subgroup of serine proteases. Serine protease is an enzyme that 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). Subtilisins preferably include, and consist of, subgroups I-S1 and I-S2 as defined by Siezen et al., Protein Engng. 4 (1991) 719-737; and Siezen et al., Protein Science 6 (1997) 501-523. Because of the highly conserved structure of the active site of serine proteases, the subtilisin according to the invention can be functionally equivalent to the proposed subgroup designated subtilase by Siezen et al. (supra).

[00042] Subtilisin can be of animal, plant or microbial origin, including chemically or genetically modified mutants (protein-manipulated variants). Examples of subtilisins are Bacillus derivatives, for example subtilisin Novo, subtilisin Carlsberg, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 (described Petition 870190104795, dated 10 / 17 / 2019, page 13 / 63 / 29 in WO 89 / 06279) and Protease PD138 (WO 93 / 18140). Examples are described in WO 98 / 020115, WO 01 / 44452, WO 01 / 58275, WO 01 / 58276, WO 03 / 006602 and WO 04 / 099401. Other examples are the variants described in WO 92 / 19729, WO 98 / 20115, WO 98 / 20116, WO 98 / 34946, WO 2011 / 036263 and mixtures of proteases.

[00043] Examples of commercially available subtilisins include Kannase™, Everlase™, Relase™, Esperase™, Alcalase™, Durazym™, Savinase™, Ovozyme™, Liquanase™, Coronase™, Polarzyme™, Pyrase™, Pancreatic Trypsin NOVO (PTN), Bio-Feed™ Pro and Clear-Lens™ Pro; Blaze (all marketed by Novozymes A / S, Bagsvaerd, Denmark). Other commercially available subtilisins include Ronozyme™ Pro, Maxatase™, Maxacal™, Maxapem™, Opticlean™, Properase™, Purafast™, Purafect™, Purafect Ox™, Purafact Prime™, Excellase™, FN2™, FN3™, and FN4™ (marketed by Genencor International Inc., Gist-Brocades, BASF, or DSM). Other examples are Primase™ and Duralase™. Blap R, Blap S, and Blap X are marketed by Henkel. Second enzyme

[00044] In addition to subtilisin, the detergent composition may optionally comprise a second enzyme such as a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a pectate lyase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, a laccase, and / or a peroxidase. The composition may contain one, two or more non-subtilisin enzymes. Lipase and cutinase

[00045] Suitable lipases and cutinases include those of bacterial or fungal origin. Chemically modified mutants or those with manipulated proteins are included. Examples include Thermomyces lipase, for example from T. lanuginosus (formerly called Humicola lanuginosa) as described in EP 258 068 and EP 305 216, and Humicola cutinase, by Petition 870190104795, dated 10 / 17 / 2019, page 14 / 63 / 29 example H. insolens as described in WO 96 / 13580, a Pseudomonas lipase, for example P. alcaligenes or P. pseudoalcaligenes (EP 218 272), P. cepacia (EP 331 376), P. stutzeri (GB 1,372,034), P. fluorescens, Pseudomonas sp. strain SD 705 (WO 95 / 06720 and WO 96 / 27002), P. wisconsinensis (WO 96 / 12012), a Bacillus lipase, for example from B. subtilis (Dartois et al., 1993, Biochemica et Biophysica Acta, 1131: 253-360), B. stearothermophilus (JP 64 / 744992) or B. pumilus (WO 91 / 16422).

[00046] Other examples are lipase variants such as those described in WO 92 / 05249, WO 94 / 01541, EP 407 225, EP 260 105, WO 95 / 35381, WO 96 / 00292, WO 95 / 30744, WO 94 / 25578, WO 95 / 14783, WO 95 / 22615, WO 97 / 04079, WO 97 / 07202, WO 00 / 060063, WO 2007 / 087508 and WO 2009 / 109500.

[00047] Commercially available lipase enzymes include Lipolase™, Lipolase Ultra™, and Lipex™; Lecitase™, Lipolex™; Lipoclean™, Lipoprime™ (Novozymes A / S). Other commercially available lipases include Lumafast (Genencor Int Inc); Lipomax (Gist-Brocades / Genencor Int Inc) and Bacillus sp. lipase from Solvay. Amylase

[00048] Suitable amylases (α and / or β) include those of bacterial or fungal origin. Chemically modified mutants or those with manipulated proteins are included. Amylases include, for example, α-amylases obtained from Bacillus, for example a special strain of Bacillus licheniformis, described in more detail in GB 1,296,839.

[00049] Examples of useful amylases are the variants described in WO 94 / 02597, WO 94 / 18314, WO 96 / 23873, and WO 97 / 43424, especially the variants with substitutions at one or more of the following positions: 15, 23, 105, 106, 124, 128, 133, 154, 156, 181, 188, 190, 197, 202, 208, 209, 243, 264, 304, 305, 391, 408, and 444. Petition 870190104795, dated 10 / 17 / 2019, page 15 / 63 / 29

[00050] Commercially available amylases are Stainzyme; Stainzyme Plus; DuramylTM, TermamylTM, Termamyl Ultra; Natalase, FungamylTM and BANTM (Novozymes A / S), RapidaseTM and PurastarTM (from Genencor International Inc.). Lyases

[00051] Pectate lyase can be a wild-type enzyme derived from Bacillus, particularly B. licherniformis or B. agaradhaerens, or a variant derived from either of these, for example as described in US 6,124,127, WO 1999 / 027083, WO 1999 / 027084, WO 2002 / 006442, WO 2002 / 092741, WO 2003 / 095638, a commercially available pectate lyase is XPect; Pectawash and Pectaway (Novozymes A / S). Mannanase

[00052] Mannanase may be an alkaline mannanase from Family 5 or 26. It may be a wild type of Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii or H. insolens. Suitable mannanases are described in WO 1999 / 064619. A commercially available mannanase is Mannaway (Novozymes A / S). Cellulase

[00053] Suitable cellulases include those of bacterial or fungal origin. Chemically modified mutants or those with manipulated proteins are included. Suitable cellulases include cellulases of the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, for example the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed in US 4,435,307, US 5,648,263, US 5,691,178, US 5,776,757 and WO 89 / 09259.

[00054] Especially suitable cellulases are alkaline or neutral cellulases with color care benefits. Examples of such cellulases are the cellulases described in EP 0 495 257, EP 0 531 372, WO 96 / 11262, WO 96 / 29397, WO 98 / 08940. Other examples are variants of cellulases such as Petition 870190104795, dated 10 / 17 / 2019, page 16 / 63 / 29 described in WO 94 / 07998, EP 0 531 315, US 5,457,046, US 5,686,593, US 5,763,254, WO 95 / 24471, WO 98 / 12307 and WO 99 / 01544.

[00055] Commercially available cellulases are Celluzyme, Celluclean; Endloase; Carezyme; Renozyme; whitezyme (Novozymes A / S). Preparation of peptide aldehyde and hydrosulfite / bisulfite adduct

[00056] Peptide aldehyde can be converted into a water-soluble hydrosulfite adduct by reaction with sodium bisulfite, as described in textbooks, for example, March, J. Advanced Organic Chemistry, fourth edition, Wiley-Interscience, US 1992, p 895.

[00057] The conversion to hydrosulfite adduct is reversible (Ex J. Am. Chem. Soc. 1978, 100, 1228). Thus, the adduct can partially or totally revert to release the peptide aldehyde in a liquid detergent, in a liquid subtilisin formulation, or in the wash water.

[00058] The peptide aldehyde in question can be prepared by known methods, for example as described in US 4,703,036, US 4,478,745 or US 5,578,574 omitting a final drying step, or it can be prepared by any of the methods reviewed in J. Pept. Sci. 2007; 13; 115 or exemplified in Synthesis 1983, 676. The peptide aldehyde can be crude or purified, isolated as a solid or held in solution by an organic solvent.

[00059] An aqueous solution of the bisulfite adduct can be prepared by reacting the corresponding peptide aldehyde with an aqueous solution of sodium bisulfite (sodium hydrogen sulfite, NaHSO3); potassium bisulfite (KHSO3) by known methods, for example, as described in WO 98 / 47523; US 6,500,802; US 5,436,229; J. Am. Chem. Soc. 1978, 100, 1228; Org. Synth., Coll. Vol. 7: 361. Detergent composition

[00060] Detergent can be liquid or granular. Liquid detergent is in a physical form that is not solid (or gaseous); it can be Petition 870190104795, dated 10 / 17 / 2019, page 17 / 63 / 29 a pourable liquid, a pourable gel or a non-pourable gel. It may be isotropic or structured, preferably isotropic. Includes formulations useful for washing in automatic washing machines or for hand washing. The detergent contains at least one surfactant. The detergent may also include an adjuvant.

[00061] The particulate detergent composition may be a granule or powder, or a powder / granule pressed into tablet or briquette form. The composition may be in tablet, bar or bag form, including bags with multiple compartments. The composition may be in powder form, for example a free-flowing powder, such as an agglomerate, dry pulverized powder, encapsulated, extruded, needle, noodle, flake or a combination thereof.

[00062] Detergent ingredients can be physically separated from each other by compartments in water-soluble bags or by layers of different soap pieces (see Unit Dose below). This can prevent a negative storage interaction between the components. Different dissolution profiles of each compartment can also lead to delayed dissolution of selected components in the washing solution.

[00063] The pouches may have any shape, form and material suitable for containing the composition, for example without allowing the composition to be released from the pouch before contact with water. The pouch is made of a water-soluble film enclosing an interior volume. This interior volume may be divided into compartments of the pouch. Preferred films are polymeric materials, preferably polymers formed into a film or sheet. Polymers, copolymers or their preferred derivatives are selected from polyacrylates, and water-soluble acrylate copolymers, methylcellulose, carboxymethylcellulose, sodium dextrin, ethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, maltodextrin, polymethacrylates, more preferably copolymers of Petition 870190104795, dated 10 / 17 / 2019, page 18 / 63 / 29 polyvinyl alcohol and hydroxypropylmethylcellulose (HPMC). Preferably, the polymer level in the film, for example PVA, is at least about 60%. The preferred average molecular weight will typically be between about 20,000 and about 150,000. The films may also be blended compositions comprising mixtures of hydrolytically degradable and water-soluble polymers such as polyantixide and polyvinyl alcohol (known by the trade reference M8630 as sold by Chris Craft In. Prod. Of Gary, Ind., US) plus plasticizers such as glycerol, ethylene glycerol, propylene glycol, sorbitol and mixtures thereof. The bags may comprise a solid laundry composition or partial components and / or a liquid laundry composition or partial components separated by a water-soluble film.The compartment for liquid components may differ in composition from the compartments containing solids (see, for example, US 2009 / 0011970).

[00064] The choice of detergent components for fabric treatment may include consideration of the type of fabric to be cleaned, the type and / or degree of soiling, the temperature at which cleaning should take place, and the detergent formulation. Although the components mentioned below are categorized by general heading according to a particular functionality, this should not be understood as a limitation, since a component may include additional functionalities, as the professional will know.

[00065] In one embodiment, the stabilized subtilisin composition of the invention can be added to a detergent composition in an amount corresponding to 0.001-100 mg of protein, such as 0.01-100 mg of protein, preferably 0.005-50 mg of protein, more preferably 0.01-25 mg of protein, even more preferably 0.05-10 mg of protein, more preferably 0.05-5 mg of protein, and even more preferably 0.01-1 mg of protein per liter of liquor. Petition 870190104795, dated 10 / 17 / 2019, page 19 / 63 / 29 washing. Unit dose

[00066] A unit-dose product is packaging containing a single dose in a non-reusable container. It is increasingly used in laundry and dishwashing detergents. A unit-dose detergent product is packaging (e.g., a pouch made of water-soluble film) containing the amount of detergent used for a single wash.

[00067] In one aspect, the detergent composition is in unit dose form. Detergent products in unit dose form include tablets, capsules, sachets, pouches, etc. In one aspect, for use herein, the tablets are wrapped with a water-soluble film and water-soluble pouches. The weight of the detergent composition of the invention is from about 10 to about 25 grams, from about 12 to about 24 grams, or even from 14 to 22 grams. These weights are extremely convenient for attaching automatic dishwashing detergent dispensers. In the case of unit dose products with water-soluble material enclosing the detergent composition, the water-soluble material is not considered part of the composition. In one aspect, the unit dose form is a water-soluble pouch (i.e., water-soluble film enclosing a detergent composition), in one aspect, a multi-compartment pouch with several films forming several compartments.This configuration contributes to the flexibility and optimization of the composition. This allows for the controlled separation and release of different ingredients. In one aspect, one compartment contains a detergent composition in solid form and another compartment contains a detergent composition in liquid form.

[00068] In one aspect, embodiments of the multi-compartment bag with two different compartments could contain two different cleaning agents. In another aspect, the films of these two compartments have different dissolution profiles, allowing the release of Petition 870190104795, dated 10 / 17 / 2019, page 20 / 63 / 29 same or different agents at different times. For example, the agent from one compartment (first compartment) may be administered at the beginning of the washing process to help remove dirt, and a second agent from another compartment (second compartment) may be administered at least two minutes, or even at least five minutes after the agent from the first compartment.

[00069] In one aspect, a multi-compartment bag is disclosed comprising two compartments side by side superimposed on another compartment in which at least two different compartments contain two different detergent compositions.

[00070] A multi-compartment package is formed by several water-soluble enveloping materials that form multiple compartments. One compartment would contain some or all of the ingredients of the detergent composition, another compartment might contain a liquid composition. The liquid composition might be aqueous (i.e., comprising more than 10% water by weight of the liquid composition), and the compartment might be made of a hot water-soluble material. In one embodiment, one compartment is made of a cold water-soluble material. This allows for the controlled separation and release of different ingredients. In other embodiments, all compartments are made of hot water-soluble material.

[00071] Suitable packages comprise at least two compartments placed side-by-side (i.e., stacked on top of) another compartment, with bags being particularly suitable. This arrangement contributes to the solidity, robustness, and strength of the package, and additionally minimizes the amount of water-soluble material required. Only three pieces of material are needed to form three compartments. The robustness of the package also allows the use of very thin films without compromising the physical integrity of the package. The package is also very easy to handle. Petition 870190104795, dated 10 / 17 / 2019, page 21 / 63 17 / 29 use because the compartments do not need to be folded for use in fixed geometry machine dispensers. At least two of the package compartments contain two different detergent compositions. By different compositions, it is meant detergent compositions that differ in at least one ingredient.

[00072] In one aspect, at least one compartment contains a solid detergent composition and another compartment an aqueous liquid detergent composition, the compositions typically having a solid-to-liquid ratio of about 20:1 to about 1:20, about 18:1 to about 2:1, or about 15:1 to about 5:1. This type of package is very versatile because it can accommodate compositions with a wide spectrum of solid:liquid ratio values. The pouches have a high solid:liquid ratio because many of the detergent ingredients are particularly well-suited for use in solid form, in a powder-form aspect. The solid:liquid ratio defined herein refers to the ratio between the weight of all solid compositions and the weight of all liquid compositions in the package.

[00073] The appropriate solid:liquid weight ratios are between about 2:1 and about 18:1, or from about 5:1 to about 15:1. These weight ratios are suitable in cases where most of the detergent ingredients are in liquid form.

[00074] In one aspect, the two compartments side by side contain liquid detergent compositions which may be the same or different and another compartment contains a solid detergent composition, for example in powder form, in one aspect, a densified powder. The solid composition contributes to the strength and robustness of the package.

[00075] For dispenser mounting reasons, especially in automatic dishwashers, the single-dose products here have a square or rectangular base and a height of between 1 and about 5 cm, or about 1 and about 4 cm. In one aspect, the weight of the composition Petition 870190104795, dated 10 / 17 / 2019, p. 22 / 63 / 29, states that the solid composition weighs approximately 5 to approximately 20 grams, or approximately 10 to approximately 15 grams, and the weight of the liquid compositions is approximately 0.5 to approximately 4 grams, or approximately 0.8 to approximately 3 grams. In one aspect, at least two of the films forming different compartments have different solubilities under the same conditions. This allows for the release at different times of the compositions that partially or totally enclose them.

[00076] Controlled release of ingredients from a multi-compartment pouch can be achieved by modifying the film thickness and / or the solubility of the film material. The solubility of the film material can be retarded, for example, by crosslinking the film as described in WO 2002 / 102955. Other water-soluble films designed for rinse-off release are described in US 4,765,916 and US 4,972,017. Waxy coating (see US 5,453,216) of films can aid rinse-off release. pH-controlled release media are described in US 5,453,216, in particular aminoacetylated polysaccharides with a selective degree of acetylation.

[00077] Other means of achieving delayed release through multi-compartment pouches with different compartments, wherein the compartments are made of films with different solubility, are disclosed in US 6,727,215. Surfactants

[00078] The detergent composition may comprise one or more surfactants, which may be anionic and / or cationic and / or non-ionic and / or semipolar and / or zwitterionic, or a mixture thereof. In a particular embodiment, the detergent composition includes a mixture of one or more non-ionic surfactants and one or more anionic surfactants, but they may also be used individually.

[00079] Surfactant(s) are typically present at a level between 0.1% and 60% by weight, such as approximately 1% to 40%, or approximately 3% to Petition 870190104795, dated 10 / 17 / 2019, p. 23 / 63 / 29 20%, or about 3% to 10%. The surfactant(s) are chosen according to the desired cleaning application, and include any conventional surfactants known in the art. Any surfactant known in the art can be used for use in detergents.

[00080] Non-limiting examples of anionic surfactants include sulfates and sulfonates, in particular, linear alkylbenzenesulfonates (LAS), LAS isomers, branched alkylbenzenesulfonates (BABS), phenylalkanesulfonates, alpha-olefin sulfonates (AOS), olefin sulfonates, alkene sulfonates, alkane-2,3-diylbis(sulfates), hydroxyalkanesulfonates and disulfonates, alkyl sulfates (AS) such as sodium dodecyl sulfate (SDS), fatty alcohol sulfates (FAS), primary alcohol sulfates (PAS), alcohol ether sulfates (AES or AEOS or FES, also known as alcohol ethoxysulfates or fatty alcohol ether sulfates, including sodium lauryl ether sulfate (SLES), soaps or fatty acids;Secondary alkanesulfonates (SAS), paraffin sulfonates (PS), ester sulfonates, glycerol sulfonated fatty acid esters, alpha-sulfo fatty acid methyl esters (alpha-SFMe or SES) including methyl ester sulfonate (MES), alkyl- or alkenylsuccinic acid, dodecenyl / tetradecenyl succinic acid (DTSA), fatty acid derivatives of amino acids, diesters and monoesters of sulfo-succinic acid or soap and combinations thereof.

[00081] When included therein, the detergent shall normally contain between about 1% and about 40% by weight, such as between about 5% and about 30%, including between about 5% and about 15%, or between about 20% and about 25% of an anionic surfactant.

[00082] Non-limiting examples of nonionic surfactants include alcohol ethoxylates (AE or AEO), alcohol propoxylates, propoxylated fatty alcohols (PFA), alkoxylated fatty acid alkyl esters, such as ethoxylated and / or propoxylated fatty acid alkyl esters, alkylphenol ethoxylates (APE), nonylphenol ethoxylates (NPE), alkyl polyglycosides (APG), amines Petition 870190104795, dated 10 / 17 / 2019, p. 24 / 63 / 29 alkoxylated, fatty acid monoethanolamides (FAM), fatty acid diethanolamides (FADA), ethoxylated fatty acid monoethanolamides (EFAM), propoxylated fatty acid monoethanolamide (PFAM), polyhydroxy alkyl fatty acid amides, or N-acyl N-alkyl glucosamine derivatives (glucamides, GA, or fatty acid glucamide, FAGA), as well as products available under the trade names SPAN and TWEEN, and combinations thereof.

[00083] When included therein, the detergent shall normally contain between about 0.2% and about 40% by weight of a non-ionic surfactant, for example between about 0.5% and about 30%, in particular between about 1% and about 20%, between about 3% and about 10%, such as between about 3% and about 5%, or from about 8% to about 12%. Adjuvants

[00084] The detergent composition may contain approximately 0-65% by weight of a detergent adjuvant or co-adjuvant or a mixture thereof. In a dishwashing detergent, the adjuvant level is typically 40-65%, in particular 50-65%. The adjuvant and / or co-adjuvant may particularly be a chelating agent that forms water-soluble complexes with Ca and Mg. Any adjuvant and / or co-adjuvant known in the art for use in laundry detergents may be used.Non-limiting examples of adjuvants include zeolites, diphosphates (pyrophosphates), triphosphates such as sodium triphosphate (STP or STPP), nitrilotriacetic acid, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, carbonates such as sodium carbonate, soluble silicates such as sodium metasilicate, layered silicates (e.g., Hoechst's SKS-6), ethanolamines such as 2-aminoethanol-1-ol (MEA), iminodiethanol (DEA) and 2,2',2"-nitrilotriethanol (TEA) and carboxymethylinulin (CMI), and combinations thereof.

[00085] The adjuvant may be a strong adjuvant such as methylglycine diacetic acid (MGDA) or N,N tetrasodium glutamic acid salt Petition 870190104795, dated 10 / 17 / 2019, page 25 / 63 / 29 Dicarboxymethyl (GLDA); it can be a medium adjuvant like sodium tripolyphosphate (STPP) or it can be a weak adjuvant like sodium citrate. Whitening system

[00086] The detergent composition may contain 0-50% by weight of a bleaching system. Any bleaching system known in the art for use in laundry detergents may be used. Suitable bleaching system components include bleaching catalysts, bleaching activators, hydrogen peroxide sources such as sodium percarbonate and sodium perborates, pre-formed peracids and mixtures thereof. Suitable pre-formed peracids include, but are not limited to, peroxycarboxylic acids and salts, percarbonic acids and salts, perimidic acids and salts, peroxymonosulfuric acids and salts, for example, Oxona (R), and mixtures thereof.Non-limiting examples of bleaching systems include peroxide-based bleaching systems, which may comprise, for example, an inorganic salt, including alkali metal salts such as sodium perborate salts (usually mono- or tetrahydrate), percarbonate, persulfate, perphosphate, persilicate salts, in combination with a bleaching activator forming a peracid. By bleaching activator is meant a compound that reacts with peroxygen lye as hydrogen peroxide to form a peracid. The peracid thus formed constitutes the activated lye.Suitable bleaching activators for use here include those belonging to the class of amide, imide or anhydride esters, suitable examples being tetraacetyl ethylenediamine (TAED), sodium sulfonate, 3,5,5-trimethylhexanoyloxybenzene, diperoxy dodecanoic acid, 4-(dodecanoyloxy)benzenesulfonate (LOBS), 4-(decanoyloxy)benzenesulfonate, 4-(decanoyloxy)benzoate (DOBS), 4-(3,5,5-trimethylhexanoyloxy)benzenesulfonate (ISONOBS), tetraacetylethylenediamine. Petition 870190104795, dated 10 / 17 / 2019, p. 26 / 63 / 29 (TAED) and 4-(nonanoyloxy)benzenesulfonate (NOBS), and / or those disclosed in WO98 / 17767. A particular family of bleaching activators of interest was disclosed in EP624154, and particularly preferred in this family is acetyltriethyl citrate (ATC). ATC or a short-chain triglyceride such as triacin has the advantage of being environmentally friendly as it eventually degrades into citric acid and alcohol. In addition, acetyltriethyl citrate and triacetin have good hydrolytic stability in the product during storage and are efficient bleaching activators. Finally, ATC provides good adjuvant capacity for laundry additives. Alternatively, the bleaching system may comprise peroxyacids, for example, of the amide, imide or sulfone type. The bleaching system may also comprise peracids such as 6-(phthaloylamino)percapronic acid (PAP).The bleaching system may also include a bleaching catalyst.

[00087] Other ingredients of the detergent composition, which are well known in the art, include hydrotropes, fabric coloring agents, antifoaming agents, soiling polymers, anti-redeposition agents, etc. Methods and Compositions

[00088] In a first aspect, the present invention provides a composition comprising a subtilisin and a peptide aldehyde hydrosulfite adduct with the formula X-B1-NH-CHR-CHOH-SO3M, wherein:

[00089] a) M is H (hydrogen) or an alkali metal;

[00090] b) R is a group in which NH-CHR-CO is an L or D amino acid residue;

[00091] c) B1 is an amino acid residue; and

[00092] d) X consists of one or more amino acid residues, optionally comprising an N-terminal protecting group.

[00093] In one embodiment, R is a group in which NH-CHR Petition 870190104795, dated 10 / 17 / 2019, p. 27 / 63 / 29 CO is an L or D amino acid residue of Tyr, m-tyrosine, 3,4-dihydroxyphenylalanine, Phe, Val, Met, Nva, Leu, Ile, or Nle.

[00094] In one embodiment, B1 is a residue of Ala, Cys, Gly, Pro, Ser, Thr, Val, Nva, or Nle.

[00095] In one embodiment, X can be B2, B3-B2, Z-B2, or Z-B3-B2, wherein B2 and B3 each represent an amino acid residue and Z is an N-terminal protecting group. Preferably, B2 is a Val, Gly, Ala, Arg, Leu, Phe, or Thr residue. Preferably, B3 is a Phe, Tyr, Trp, phenylglycine, Leu, Val, Nva, Nle, or Ile residue.

[00096] In one embodiment, Z is benzyloxycarbonyl (Cbz), p-methoxybenzyl carbonyl (MOZ), benzyl (Bn), benzoyl (Bz), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), formyl, acetyl (Ab), methyloxy or methyloxycarbonyl.

[00097] In one embodiment, the composition is in liquid or granular form. Preferably, the composition is a detergent further comprising a surfactant.

[00098] In one embodiment, the composition further comprises a second enzyme, particularly a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a pectate lyase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, a laccase or a peroxidase.

[00099] In another aspect, the invention provides a method for preparing the composition of the invention, as described above, a method comprising mixing a subtilisin, an aqueous solution comprising a peptide aldehyde hydrosulfite adduct with the formula X-B1-NH-CHRCHOH-SO3M, as described above, and optionally a surfactant. [000100] In yet another aspect, the invention provides a compound for use in the composition of the invention, a compound that is a hydrosulfite aldehyde peptide adduct with the formula X-B1-NH-CHR-CHOH. [Illegible text: 870190104795, 10 / 17 / 2019, page 28 / 63 / 29] SO3M, where M and X are defined as described above, B1 is an amino acid residue other than proline (Pro), and R is a group in which NH-CHR-CO is an L or D amino acid residue of Tyr, m-tyrosine, 3,4-dihydroxyphenylalanine, Phe, Val, Met, Nva, or Nle. [000101] In one embodiment the compound is Cbz-RANHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, benzyloxycarbonyl. Ac-GACbz-GACbz-GGCbz-RVCbz-LVAc-LGAAc-FGAAc-YGAAc-FGVou Ac-WLVem where Ac is acetyl and Cbz is [000102] In yet another aspect, the invention provides a compound for use in the composition of the invention, which is a peptide aldehyde hydrosulfite adduct with the formula X-B1-NH-CHR-CHOHSO3M, wherein M, R and X are defined as described above and wherein B1 is an amino acid residue of alanine (Ala), cysteine ​​(Cys), glycine (GIy), serine (Ser), threonine (Thr), valine (VaI), norvaline (Nva) and norleucine (Nle). [000103] In one embodiment the compound is Cbz-GANHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, Cbz-VANHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, Cbz-GANHCH(CH2Ph)C(OH)(SO3M)-H, Cbz-GA-NHCH(CH(CH3)2)C(OH)(SO3M)H, Cbz-GG-NHCH(CH2Ph)C(OH)(SO3M)-H, Ac-FGANHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, Ac-FGA. Petition 870190104795, 10 / 17 / 2019, page 29 / 63 / 29 NHCH(CH2Ph)C(OH)(SÜ3M)-H, Ac-FGA-NHCH(CH2CH2SCH3)(SÜ3M)-H, MeO-CO-VA-NHCH(CH2CH(CH3)2))C(OH)(SÜ3M)-H, MeNCO-VANHCH(CH2CH(CH3)2))C(OH)(SÜ3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2Ph)C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, MeO-CO-FGAMeO-CO-FGAMeSO2-FGAMeSO2-VAPhCH2O(OH)(O)P-VAEtSO2-FGAPhCH2SO2-VAPhCH2O(OH)(O)P-LAPhCH2O(OH)(O)P-FAe MeO(OH)(O)P-LGANHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, em que Ac é acetila e Cbz é benziloxicarbonila. [000104] In yet another aspect, the invention provides a compound for use in the composition of the invention, which compound is a peptide aldehyde hydrosulfite adduct with the formula X-B1-NH-CHR-CHOHSO3M; where R and M are defined as described above; B1 represents an amino acid residue that is different from proline (Pro); X is B2, B3-B2, Z-B2, ZB3-B2, where B3 and B2 each represent an amino acid residue, and Z is an N-terminal protecting group; and where B2 is Gly, Thr or Val. [000105] In yet another aspect, the invention provides a compound for use in the composition of the invention, which compound is a peptide aldehyde hydrosulfite adduct with the formula X-B1-NH-CHR-CHOHSO3M; where R and M are defined as described above; B1 represents an amino acid residue that is different from proline (Pro); X is B3-B2, Z-B3-B2, where B3 and B2 each represent an amino acid residue, and Z is an N-terminal protecting group; and where B3 is Phe, Tyr, Trp, phenylglycine, Leu, Val, Nva, Nle or Ile. Petition 870190104795, dated 10 / 17 / 2019, page 30 / 63 / 29 EXAMPLES [000106] Chemicals used as buffers and substrates were commercial products of at least reagent grade. EXAMPLE 1 Preparation of a hydrosulfite adduct of a peptide aldehyde. [000107] A peptide aldehyde with the formula X-B1-B0-H, as described above, is converted into the corresponding bisulfite adduct. To a stirred suspension of 1 mmol X-B1-B0-H in 8 ml of ethyl acetate at room temperature, 1.06 mmol of sodium bisulfite (114 mg) in 6 ml of water is added dropwise, and the reaction is stirred for 2 hours. The aqueous phase is isolated, and the organic layer is washed with 2 x 2 ml of water. The combined aqueous phases are lyophilized, providing X-B1-NH-CHR-CHOH-SO3Na (expected yield of 66%) as a white powder. EXAMPLE 2 Preparation of a liquid preparation of a hydrosulfite adduct of a peptide aldehyde. [000108] 4 mmol of solid X-B1-NH-CHR-CHOH-SO3Na are dissolved in 6 g of demineralized water and stirred for 30 min at 35°C and subsequently cooled to provide a 25% aqueous solution of X-B1NH-CHR-CHOH-SO3Na. Alternatively, the aqueous phase of the above synthesis could be readily used for enzymatic stabilization in the next step and this solution is approximately 3% (weight percent). EXAMPLE 3 Preparation of a stabilized subtilisin formulation comprising subtilisin and a hydrosulfite aldehyde peptide adduct. [000109] To a commercially available Savinase 16L™ (Novozymes A / S, Bagsvaerd, Denmark) is added 0.9% X-B1-NH-CHRCHOH-SO3Na using 3.6% of the above 25% aqueous solution. EXAMPLE 4 Petition 870190104795, dated 10 / 17 / 2019, page 31 / 63 / 29 Stabilization of subtilisin by hydrosulfite adduct [000110] General details of the experiment: Detergents containing a subtilisin (Savinase 16 L) and a lipase (Lipex 100 L) with or without X-B1-NHCHR-CHOH-SO3Na (the aldehyde peptide hydrosulfite adduct can be added separately from the detergent containing the subtilisin) are placed in sealed glass containers at -18°C, 35°C, and 40°C. Residual protease and lipase activities are measured at different times using standard analytical methods (protease by hydrolysis of N,N-dimethylcasein at 40°C, pH 8.3 and lipase by hydrolysis of pNp-valerate at 40°C, pH 7.7). Similar assays can be performed by adding subtilisin to a detergent already containing adequate amounts of the aldehyde peptide hydrosulfite adduct. Similar results can be obtained with all the aldehyde peptide bisulfite adducts mentioned. [000111] The composition described in Table 1 was made as an example of a detergent composition with a stabilized subtilisin formulation. Table 1. Detergent composition. Component % (percentage by weight) Sodium alkylethoxy sulfate (C9-15, 2EO) 6.0 Sodium dodecylbenzenesulfonate 3.0 Sodium toluene sulfonate 3.0 Oleic acid 2.0 Primary alcohol ethoxylate (C12-15, 7EO) 3.0 Primary alcohol ethoxylate (C12-15, 3EO) 2.5 Ethanol 0.5 Monopropylene glycol 2.0 Trisodium citrate 2H2O 4.0 Triethanolamine 0.4 pH adjusted to 8.5 with NaOH Savinase formulation stabilized with X-B1-NH-CHR-CHOH-SO3Na; or Savinase 16 L for comparison 0.5 Lipex 100 L™ (marketed by Novozymes A / S) 0.5 100% water [000112] Table 2 shows the results obtained with X= CbzGly; B1= Ala; R=-CH2-p (C6H4)-OH; stability measured after one week of incubation at 40°C in the detergent described in Table 1. Petition 870190104795, dated 10 / 17 / 2019, page 32 / 63 / 29 Table 2. Remaining protease activity after one week of incubation at 40°C in the detergent described in Table 1. Amount of inhibitor added to the detergent Protease stability (remaining activity) none 12% 7 ppm 52% 14 ppm 77% 18 ppm 80% [000113] The results demonstrate stabilization of subtilisin by the addition of a hydrosulfite adduct to a peptide aldehyde. EXAMPLE 5 Stabilization of subtilisin and lipase [000114] The composition described in Table 3 was made as an example of a detergent composition with a stabilized subtilisin formulation. Table 3. Detergent composition. Component % (percentage by weight) Sodium dodecylbenzenesulfonate 6.0 NaOH 1.4 Soybean fatty acid (Edenor SJ) 3.0 Coconut fatty acid (Radiacid 0631) 2.5 Primary alcohol ethoxylate (C13, 8EO) 5.0 Ethanol 5.0 Monopropylene glycol 5.0 Trisodium citrate 2H2O 0.5 Triethanolamine 2.0 Phosphonate - Dequest 2066 C2 3.0 pH adjusted to 8.4 with additional NaOH if necessary Subtilisin: Savinase, stabilized with X-B1-NH-CHR-CHOH-SO3Na; or with 4-formylphenyl boronic acid; or without stabilizer for comparison 0.75 Lipase: Lipex 100 L™ (marketed by Novozymes A / S) 0.15 100% Water [000115] Table 4 shows results measured after two weeks of incubation at 35°C in the detergent described in Table 3. Table 4. Remaining activity of Subtilisin (protease) and Lipase after 2 weeks of incubation at 35°C in the detergent described in Table 3. Detergent inhibitor Subtilisin: Lipase none - 2% 1% boronic acid 4-formylphenyl (prior art) 128 ppm 8% 2% X = Cbz-Gly; B1 = Ala; R = -CH2- p (C6H4)-OH 21 ppm 63% 18% Petition 870190104795, dated 10 / 17 / 2019, page 33 / 63 / 29 [000116] The results demonstrate that the addition of a peptide aldehyde hydrosulfite adduct can stabilize subtilisin and the second enzyme (lipase), and that it is much more effective than the stabilizer of the prior art. EXAMPLE 6 Stabilization of subtilisin and lipase [000117] The composition described in Table 5 was made as yet another example of a detergent with a stabilized subtilisin formulation. Table 5. Detergent composition. Component % (percentage by weight) Commercial non-enzymatic detergent. Liquid EU 2x doses. Acquired in the United Kingdom in 2009 98.2 Subtilisin: 1.4 Lipase: Lipoclean 2000L (marketed by Novozymes A / S) 0.4 [000118] Table 6 shows results measured after four weeks of incubation at 37°C and after 8 weeks of incubation at 30°C in the detergent described in Table 5. Table 6. Remaining Subtilisin (protease) and Lipase activity after 4 weeks of incubation at 37°C or after 8 weeks of incubation at 30°C in the detergent described in Table 5. Subtilisin Inhibitor: Amount of inhibitor added to detergent Subtilisin: Lipase 4 weeks at 37°C 8 weeks at 30°C none Coronase 48L 0 69% 28% X = Cbz-Gly; B1 = Ala; R = -CH2- p (C6H4)-OH Coronase 48L 59 ppm 80% 61% none Liquanase 2.5L 0 43% 0% X = Cbz-Gly; B1 = Ala; R = -CH2- p (C6H4)-OH Liquanase 2.5L 34 ppm 83% 27% [000119] The results confirm that the addition of a hydrosulfite aldehyde peptide adduct can stabilize subtilisin and the second enzyme (lipase) for several subtilisins.

Claims

CLAIM 1. Composition, characterized in that it comprises a subtilisin and a peptide aldehyde hydrosulfite adduct with the formula X-B1NH-CHR-CHOH-SO3M, wherein: a) M is H (hydrogen) or an alkali metal; b) R is a group in which NH-CHR-CO is an L or D amino acid residue; c) B1 is an amino acid residue; d) X consists of one or more amino acid residues, optionally comprising an N-terminal protecting group.

2. Composition according to claim 1, characterized in that R is a group in which NH-CHR-CO is an L or D amino acid residue of Tyr, m-tyrosine, 3,4-dihydroxyphenylalanine, Phe, Val, Met, Nva, Leu, Ile or Nle.

3. Composition according to claim 1 or 2, characterized in that B1 is a residue of Ala, Cys, Gly, Pro, Ser, Thr, Val, Nva or Nle.

4. Composition according to any one of claims 1 to 3, characterized in that X is B2, B3-B2, Z-B2, or ZB3-B2, wherein B2 and B3 each represent an amino acid residue and Z is an N-terminal protecting group.

5. Composition according to claim 4, characterized in that B2 is a residue of Val, Gly, Ala, Arg, Leu, Phe or Thr.

6. Composition according to claim 4, characterized in that B3 is a residue of Phe, Tyr, Trp, phenylglycine, Leu, Val, Nva, Nle or Ile.

7. Composition according to any one of claims 1 to 6, characterized in that Z is benzyloxycarbonyl (Cbz), p-methoxybenzyl carbonyl (MOZ), benzyl (Bn), benzoyl (Bz), p-methoxybenzyl (PMB), p-methoxyphenyl (PMP), formyl, acetyl (Ab), methyloxy or methyloxycarbonyl.

8. Composition according to any one of claims 1 to 7, characterized in that it is in liquid or granular form.

9. Composition according to any one of claims 1 to 8, characterized in that it is a detergent comprising a surfactant.

10. Composition according to any one of claims 1 to 9, characterized in that it further comprises a second enzyme, particularly a lipase, a cutinase, an amylase, a carbohydrase, a cellulase, a pectinase, a pectate lyase, a mannanase, an arabinase, a galactanase, a xylanase, an oxidase, a laccase, or a peroxidase.

11. Method for preparing the composition defined in any one of claims 1 to 10, characterized in that it comprises mixing a subtilisin, an aqueous solution comprising a hydrosulfite aldehyde peptide adduct with the formula X-B1-NH-CHRCHOH-SO3M, wherein M, R, B1 and X are as defined in claim 1, and optionally a surfactant.

12. Compound for use in the composition defined in any one of claims 1 to 10, characterized in that it is a hydrosulfite aldehyde peptide adduct with the formula X-B1-NH-CHRCHOH-SO3M, wherein M and X are defined in claim 1, B1 is an amino acid residue other than proline (Pro) and R is a group in which NHCHR-CO is an L or D amino acid residue of Tyr, m-tyrosine, 3,4-dihydroxyphenylalanine, Phe, Val, Met, Nva or Nle.

13. Compound according to claim 12, characterized in that it is Cbz-RA-NHCH(CH2C6H4OH)C(OH)(SO3M)-H, Ac-GA- Petition 870190104795, dated 10 / 17 / 2019, page. 36 / 63 3 / 4 NHCH(CH2C6H4OH)C(OH)(SÜ3M)-H, NHCH(CH2C6H4OH)C(OH)(SÜ3M)-H, NHCH(CH2C6H4ÜH)C(OH)(SÜ3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, NHCH(CH2C6H4OH)C(OH)(SO3M)-H, benzyloxycarbonyl. Cbz-GACbz-GGCbz-RVCbz-LVAc-LGAAc-FGAAc-YGAAc-FGVou Ac-WLVem where Ac is acetyl and Cbz is 14. Compound for use in the composition defined in any one of claims 1 to 10, characterized in that it is a hydrosulfite aldehyde peptide adduct with the formula X-B1-NH-CHRCHOH-SO3M, wherein M, R and X are defined as in claim 1 and wherein B1 is an amino acid residue of alanine (Ala), cysteine ​​(Cys), glycine (Gly), serine (Ser), threonine (Thr), valine (Vai), norvaline (Nva) and norleucine (Nle).

15. The compound of claim 14, characterized by being Cbz-GA-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, CbzVA-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, Cbz-GA-H NHCH(CH2Ph)C(OH)(SO3M)-H, Cbz-GA-NHCH(CH(CH3)2)C(OH)(SO3M)H, Cbz-GG-NHCH(CH2Ph)C(OH)(SO3M)-H, Ac-FGANHCH(CH2CH(CH3)2))CMOH-(SO3M)-H Ac-FGANHCH(CH2Ph)C(OH)(SO3M)-H, Ac-FGA-NHCH(CH2CH2SCH3)(SO3M)-H, MeO-CO-VA-NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, MeNCO-VANHCH(CH22)(CH22)(SO3M)(SO3M)-H MeO-CO-FGANHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, MeO-CO-FGANHCH(CH2Ph)C(OH)(SO3M)-H, MeSO2-FGAPetition 870190104795, of 17 / 10 / 2019, p. 37 / 63 4 / 4 NHCH(CH2CH(CH3)2))C(OH)(SÜ3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SÜ3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3))(SO3M)-H NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, NHCH(CH2CH(CH3)2))C(OH)(SO3M)-H, MeSO2-VAPhCH2O(OH)(O)P-VAEtSO2-FGAPhCH2SO2-VAPhCH2O(OH)(O)P-LAPhCH2O(OH)(O)P-FAe MeO(OH)(O)P-LGANHCH(CH2CH(CH3)2))C(OH)(SO3M) is Ace and C-H benzyloxycarbonyl.

16. Compound for use in the composition defined in any one of claims 1 to 10, characterized in that it is a peptide aldehyde hydrosulfite adduct with the formula X-B1-NH-CHRCHOH-SO3M; where R and M are defined as in claim 1; B1 represents an amino acid residue that is different from proline (Pro); X is B2, B3-B2, Z-B2, Z-B3-B2, where B3 and B2 each represent an amino acid residue, and Z is an N-terminal protecting group; and where B2 is Gly, Thr or Val.

17. Compound for use in the composition defined in any one of claims 1 to 10, characterized in that it is a hydrosulfite aldehyde peptide adduct with the formula X-B1-NH-CHRCHOH-SO3M; where R and M are defined as in claim 1; B1 represents an amino acid residue that is different from proline (Pro); X is B3-B2, Z-B3-B2, where B3 and B2 each represent an amino acid residue, and Z is an N-terminal protecting group; and where B3 is Phe, Tyr, Trp, phenylglycine, Leu, Val, Nva, Nle or Ile.