Stabilised liquid enzyme compositions

FIELD: chemistry.

SUBSTANCE: invention relates to peptide aldehydes with tyrosine as C-end residue, which are especially efficient for stabilization of subtilisin-type proteases in liquid detergents.

EFFECT: obtaining novel composition.

6 cl, 3 ex

 

The technical FIELD

The present invention relates to liquid compositions, including subtilisin and peptide compound as a stabilizer for subtilisin. The invention also relates to a peptide compound that is used as a stabilizer for subtilisin.

The LEVEL of TECHNOLOGY

Protease type subtilisin well known among liquid water detergents, especially to use for washing in the Laundry. A common problem in such liquid detergents is a decomposition of the subtilisin other enzymes in the composition and decomposition of the subtilisin. Therefore, the stability of subtilisin and other enzymes in the liquid detergent composition is lowered, resulting in obtaining a liquid detergent with a low degree of efficiency of washing.

In the documents of the prior art discussed actively improve the stability during storage of enzymes in liquid detergents, for example, by the addition of various inhibitors or stabilizers subtilisin. It is known that boric acid and boranova acid reversibly inhibit proteolytic enzymes. The use of peptide aldehydes in the liquid detergents are disclosed in WO 94/04651, WO 98/13458, WO 98/13459, WO 98/13460 and WO 98/13462. More specifically, WO94/04651 discloses the use of a peptide aldehyde is in, Phe-Gly-Ala-PheH and Phe-Gly-Ala-LeuH, to stabilize proteases type subtilisin. WO94/04651 also reveals Leu-Leu-TyrH as a suitable peptide aldehyde to stabilize proteases type of chymotrypsin. In addition, WO94/04651 offers methylcarbamate or metallocene as N-terminal protective group of the peptide aldehydes. WO98/13460 discloses the use of peptide inhibitors of proteases and peptide aldehydes and triftormetilfosfinov, where the peptide chain contains 2-5 amino acids, and aldehyde/triptorelin derived from the amino acids alanine, valine, isoleucine, leucine, phenylglycine, phenylalanine or homophenylalanine and where the N-terminal protective group preferably represents a sulfonamide or amidophosphate.

WO2007/141736, WO2007/145963 and WO2007/145964 disclose the use of reversible peptide inhibitors of proteases to stabilize the compositions of liquid detergents. US2003/157088 describes compositions containing enzymes, stable inhibitors.

WO 96/41638 and WO 2005/105826 reveal aldehydes and ketones peptides.

The INVENTION

The inventors have unexpectedly discovered that certain aldehyde or ketone derivatives of the peptides are particularly effective for the stabilization of the protease type subtilisin in aqueous compositions, such as liquid detergents, including peptide compounds with OH-substituted F. what nylalanine as the C-terminal residue.

Accordingly, the invention features a liquid composition that includes subtilisin and peptide compound of formula B2-B1-B0-R

R represents hydrogen, CH3CX3, CHX2or CH2X, where X represents a halogen atom; and

- B1represents one amino acid residue.

B0may be a phenylalanine residue with an OH substituent in thepair-position and/or inmeta-position; and (B2may consist of one or more amino acid residues, and B2optionally includes the N-terminal protective group. Alternatively, B0can be a single amino acid residue; and B2represents the residue of Gly, Arg or Leu with attached N-terminal protecting group.

The invention further provides a peptide compound of formula B2-B1-B0-R

R represents hydrogen, CH3CX3, CHX2or CH2X, where X represents a halogen atom; and

- B1represents one amino acid residue.

- B0may be a phenylalanine residue with an OH substituent in thepair-position and/or inmeta-position; and (B2may consist of one or more amino acid residues with benzyloxycarbonyl as N-terminal for itoi group, or B2can represent the residue of Gly, Arg or Leu with attached N-terminal protecting group. Alternatively, B0can be a single amino acid residue, and B1can be a small amino acid residue, and B2can represent the residue of Gly, Arg or Leu with an attached aromatic N-terminal protecting group.

DETAILED description of the INVENTION

Definition

"Amino acid residue" means a structure that is similar to the-NH-CHR-CO-, with the designation of the N-Terminus on the left and C-Terminus on the right.

The names of the amino acid residues are reduced by using single-letter or three-letter abbreviations, including the following abbreviations: alanine (A), phenylalanine (F), glycine (G), leucine (L), arginine (R), valine (V), tryptophan (W), tyrosine (Y). The abbreviation "Y-H" denotes tyrosinase, implying that the C-terminal region of tyrosine residue transformed from a carboxyl group and aldehyde group. Tyrosinase can be obtained using known methods.

Amino acids

Each amino acid residue in the B1and B2can be a natural or synthetic alpha - or beta-amino acids containing the structure-NH-(CH(R))n-C(=O)-, where n=1-2 (preferably 1), and R is selected from linear or branched and/or cyclic, substituted or nezametno the structure of the following groups: C 1-C6alkyl; phenyl; C7-C9alkylaryl; C4-C8cycloalkyl. This includes both L - and D-forms of amino acids.

The amino acid may be an alpha amino acid, such as any of the natural amino acids, Norvaline (Nva), norleucine (Nle), homophenylalanine (Hph) or phenylglycine (Pgl). Alpha-carbon atom of the amino acids can be represented in D - or L-configuration.

Peptide compound

OH-substituted phenylalanine, such as tyrosine, is a relatively hydrophilic amino acid, and its presence in the peptide will generally increase the solubility of the peptide compared to the more hydrophobic amino acids type of phenylalanine, leucine, alanine, cysteine, isoleucine, methionine and valine, all of which have a positive index of hydrophobicity compared with a negative index of hydrophobicity tyrosine (Kyte&Doolittle (1982), J. Mol. Biol. 157 (1), pp 105-132) (the higher the index of hydrophobicity, the more hydrophobic amino acid).

Peptide compound can have the formula:

where R represents hydrogen, CH3CX3, CHX2or CH2X, where X represents a halogen atom; X' represents OH or H, and at least one X' represents OH; B1represents one amino acid residue; and B2pre is is composed of one or more amino acid residues, and B2optionally includes the N-terminal protective group.

Thus, B0(amino acid residue at the C-end) can be a tyrosine residue (p-tyrosine),m-tyrosine or 3,4-dihydroxyphenylalanine. Together with tyrosinosis residue peptide compound has the following formula:

In one particular aspect of the invention, the peptide compound comprises only 3 amino acid residue, including C-terminal residue. In this aspect of the invention, the synthesis is more cost effective, and recognized that the compounds are highly effective inhibitors of enzyme activity. Preferably, the peptide compounds containing only three amino acid residue, a protected N-terminal protecting group. Accordingly, in this aspect, the invention relates to compounds where B2represents one amino acid residue comprising N-terminal protective group.

In a preferred aspect of the invention, the peptide compound is an aldehyde, comprising only 3 amino acid residue, where B2choose from arginine, glycine and leucine, including N-terminal protective group. In the case where the peptide compound is an aldehyde, comprising only 3 amino acid residue, B2prefer the Ino selected from arginine and glycine, including N-terminal protective group.

In another aspect of the invention, the peptide compound comprises at least four amino acid residue. Preferably, the peptide compound containing at least four amino acid residue, protects with N-terminal protective group. Accordingly, in this aspect, the invention relates to compounds where B2represents at least two amino acid residue comprising N-terminal protective group.

In a preferred aspect, in the case where the peptide compound comprises at least four amino acid residue, B2includes N-terminal amino acid residue containing non-polar side chain. In a more preferred embodiment, the second amino acid residue of the B2counting from the point of attachment to B1contains non-polar side chain. In an even more preferred embodiment the peptide compound comprises four amino acid residue, N-terminal amino acid residue containing non-polar side chain, selected from glycine, leucine, phenylalanine, tyrosine and tryptophan. Preferably, N-terminal amino acid residue further includes N-terminal protective group.

Preferably, B1was a small amino acid residue. More predpochtitel is but B1is an alanine or valine. In this context, it is assumed that the following amino acids are small amino acids: alanine, cysteine, glycine, Proline, serine, threonine, valine, Norvaline, norleucine.

Peptide compound may be an aldehyde, where R represents hydrogen, B1represents one amino acid, preferably selected from small amino acids such as valine and alanine, B2includes at least two amino acid residue, and where at least one of these amino acid residues are selected from phenylalanine, glycine and leucine, and where the second amino acid residue of the B2containing non-polar side chain, selected from phenylalanine, glycine, leucine, tyrosine and tryptophan. Preferably, B2include acetyl (Ac) N-terminal protective group, representing, inter alia, compounds of peptide aldehydes Ac-FGAY-H, Ac-LGAY-H, Ac-YGAY-H, Ac-FGVY-H and Ac-WLVY-H. Preferably, the compounds according to this aspect of the invention include less than 10 amino acid residues, such as 9, 8, 7, 6, 5, or, most preferably 4 amino acid residue.

In another aspect, the peptide compound can be tripeptidyl aldehyde, where R represents hydrogen, B1represents one amino acid residue selected the C small amino acids, for example, valine and alanine, B2includes amino acid residues selected from arginine, glycine and leucine. Preferably, B2includes N-terminal protective group selected from benzyloxycarbonyl (Z) and acetyl (Ac), with inter alia compounds of peptide aldehydes Z-RAY-H, Z-GAY-H, Z-GAL-H, Z-GAF-H, Z-GAV-H, Z-RVY-H, Z-LVY-H and Ac-GAY-H. Most preferably, according to this aspect of B2includes benzyloxycarbonyl (Z) N-terminal protective group.

In a preferred aspect, in the case where the peptide compound comprises at least four amino acid residue, B2includes N-terminal amino acid residue containing non-polar side chain. In the context of the present invention using the term "amino acids with nonpolar side chains" refers to amino acid or amino acid residue selected from the group including phenylalanine, tyrosine, tryptophan, isoleucine, leucine, methionine, valine, alanine, Proline, Norvaline or norleucine.

Particularly preferred peptide aldehydes of the present invention include Z-RAY-H, Ac-GAY-H, Z-GAY-H, Z-GAL-H, Z-GAF-H, Z-GAV-H, Z-RVY-H, Z-LVY-H, Ac-LGAY-H, Ac-FGAY-H, Ac-YGAY-H, Ac-FGVY-H or Ac-WLVY-H, where Z is benzyloxycarbonyl and Ac represents acetyl.

N-terminal protective group

N-terminal protective group can be any aminobenzene protection is a second group, which can be used in peptide synthesis. Publication Gross and Meinhoffer, eds., The Peptides, Vol. 3; 3-88 (1981), Academic Press, New York 1981 discloses numerous suitable protective group for the amine and for this purpose are incorporated herein by reference.

Examples of suitable groups include formyl, acetyl, benzoyloxy, trifluoracetyl, formicoxenini, methoxystyrene, aromatic urethane protective group, such as benzyloxycarbonyl; and aliphatic urethane protective groups such as tert-butyloxycarbonyl or adamantanecarbonyl, para-methoxybenzylamine (MOZ), benzyl (Bn), para-methoxybenzyl (PMB) or pair-metoksifenilny (PMP).

Preferably, N-terminal protective group of the present invention are selected from formyl, acetyl, benzoyl, aromatic or aliphatic urethanes, more preferably acetyl or benzyloxycarbonyl. In the case where the peptide compound comprises three amino acids, N-terminal protective group preferably represents an aromatic or aliphatic urethane urethane or aromatic N-terminal protective group, in particular, benzyloxycarbonyl (Cbz), para-methoxybenzylamine (MOZ), benzyl (Bn), para-methoxybenzyl (PMB) or para-methoxyphenyl (PMP), more preferably, benzyloxycarbonyl. In the case where Pat is the initial connection includes four or more amino acids, preferably, N-terminal protective group represented formyl, acetyl or benzoyl, more preferably acetyl.

Liquid composition

In the preferred embodiment of the peptide compounds of the present invention are used for stabilization or inhibition of subtilisin in liquid compositions, which may optionally include a surfactant or other enzymes.

In one aspect the invention additionally relates to the use of the compounds defined above, to stabilize and/or inhibition of enzymes, including protease type subtilisin. In a preferred aspect, the enzymes are stabilized and/or inhibited in liquid detergents. The addition of peptide compounds for liquid cleaning medium can increase the washing capacity.

The liquid composition may be an enzyme composition that includes subtilisin and optionally a second enzyme. The second enzyme may be any commercially available enzyme, in particular an enzyme selected from the group consisting of proteases, amylases, lipases, cellulases, mannanase, oxidoreductases, LiAZ and any mixture. It also includes a mixture of enzymes from the same class (e.g., proteases). The enzyme composition may also include other stabilizers, for example, polio is, such as glycerin or propylene glycol, for example, in the amount of 25-75% by weight.

The amount of enzyme used in the liquid composition varies according to the type of enzyme(s) and type of composition. In the composition, such as liquid detergent, the amount of each enzyme is designed for pure enzyme protein, as a rule, is 0.04 to 80 μm, in particular 0.2 to 30 μm, in particular 0.4 to 20 μm (typically, 1-2000 mg/l, in particular, 5-750 mg/l, in particular, 10-500 mg/l). In the composition, such as an enzyme concentrate, the amount of each enzyme is designed for pure enzyme protein, as a rule, is 0.01-20 mm, in particular 0.04 to 10 mm, in particular 0.1 to 5 mm (typically 0.3 to 500 g/l, in particular, 1-300 g/l, in particular, 3-150 g/l).

Enzymes are normally incorporated into the detergent composition sufficient to obtain the cleaning effect level, which is known to the person skilled in the art. Usually the number of enzymes is in the range from 0.0001% (wt./mass.) up to 5% (wt./mass.). Typical amounts are in the range from 0.01% to 1% by weight of the liquid detergent composition. The molar ratio of the stabilizer or inhibitor of the enzyme according to the invention the protease is at least 1:1 or 1.5:1, and is less than 1000:1, more preferably less than 500:1, more preferably from 100:1 to 2:1 or 20:1 to 21, or, most preferably, the molar ratio is from 10:1 to 3:1.

In one preferred aspect of the invention relates to compositions comprising from 1 to 95% of detergents(it) surfactant(CSOs) substances(a) by weight, from 0.0001 to 5% by weight of subtilisin, and from 0.00001 to 1% of the inhibitor peptide mass defined above. In a more preferred embodiment the invention relates to compositions comprising from 2 to 60% by weight of detergent(it) surfactant(CSOs) substances(a), from 0.0005 to 1% of subtilisin by weight, and from 0,00005 to 0.2% defined above inhibitor peptide mass. In an even more preferred embodiment the invention relates to compositions comprising from 3 to 50% detergent(it) surfactant(CSOs) substances(a) by weight, from 0.001 to 0.5% of subtilisin by weight, and from 0.0001 to 0.1% defined above inhibitor peptide mass.

Subtilisin

Subtilisin can be animal, vegetable or microbial origin, including chemically or genetically modified mutants. He may be a serine protease, preferably an alkaline microbial protease. Examples are protease type subtilisin from groups I-S defined by Siezen et al. (Protein Engineering, 1991, vol. 4 no. 7 pp. 719-737). Examples of subtilisins are subtilisin derived fromBacillusfor example, subtilisin Novo, subtilisin Carlsberg, subtilisin BPN', subtilisin 309, subtilisin 147 and subtilisin 168 (described in WO 89/06279). The examples described in WO 1998/020115, WO 01/44452, WO 01/58275, WO 01/58276, WO 2003/006602 and WO 2004/099401.

Examples of commercially available proteases (peptidases) include KannaseTM, EverlaseTM, EsperaseTM, AlcalaseTM, NeutraseTM, DurazymTM, SavinaseTM, OvozymeTM, LiguanaseTM, PolarzymeTM, PyraseTM, pancreatic Trypsin NOVO (PTN), BiO-FeedTMPro and Clear-LensTMPro (all available from Novozymes A/S, Bagsvaerd, Denmark). Other commercially available proteases include RonozymeTMPro, MaxataseTM, MaxacalTM, MaxapemTM, OpticleanTM, ProperaseTM, PurafectTM, Purafect OxTMand Purafect PrimeTM(available from Genencor International Inc., Gist-Brocades, BASF, or from DSM Nutritional Products).

The second enzyme

In addition to the subtilisin liquid composition may include a second enzyme selected from the group consisting of amylases, lipases, cellulases, mannanase, oxidoreductases and LiAZ; especially preferred is a liquid composition in which the second enzyme is a lipase.

Suitable amylases (alpha and/or beta) include amylase of bacterial or fungal origin. This includes chemically or genetically modified mutants. Amylases include, for example, alpha-amylase fromB.licheniformisdescribed in GB 1296839. Commercially available amylases are Durayl TM, TermamylTM, StainzymeTM, Stainzyme PlusTM, Termamyl UltraTM, FungamylTMand BANTM(available from Novozymes A/S), RapidaseTM, Maxamyl PTM, Purastar and Purastar OxAm (available from Gist-Brocades and Genencor Inc.).

Suitable cellulase can be bacterial or fungal origin. This includes chemically or genetically modified mutants. Cellulase can be fromHumicola insolens(US 4435307) or fromTrichodermafor exampleT.reeseiorT.viride. Examples of cellulases described in EP 0495257. Commercially available cellulase include CarezymeTM, CelluzymeTM, CellucleanTM, CelluclastTMand EndolaseTM(available from Novozymes), Puradax, Puradax HA and Puradax EG (available from Genencor).

Suitable oxidoreductase include peroxidase or oxidase, such as laccase. This includes chemically or genetically modified mutants. The peroxidase may be of plant, bacterial or fungal origin. Examples are a peroxidase derived from a strain ofCoprinusfor example, C.cineriusor C.macrorhizusor from a strain ofBacillusfor example,B. pumilusin particular, the peroxidase according to WO 91/05858. Suitable laccase according to the present document include laccase bacterial or fungal origin. Examples are a laccase fromTrametesfor example,T.villosaorT.versicoloror from strain<> Coprinusfor example,C.cinereusor from a strain ofMyceliophthorafor example,M.thermophila.

Suitable lipolytic enzymes include lipase or cutinase bacterial or fungal origin. This includes chemically or genetically modified mutants. Examples include lipase fromThermomyces lanuginosus(Humicola lanuginosa), described in EP 258068 and EP 305216, lipaseRhizomucor mieheidescribed, for example, in EP 238023, lipaseCandidasuch as lipaseC.antarcticafor example, lipase a or b fromC.antarcticathat described in EP 214761, lipaseFusarium oxysporum(WO 98/26057), lipasePseudomonassuch as lipase fromP.pseudoalcaligenesandP.alcaligenesthey are described , for example, in EP 218272, lipaseP.cepaciadescribed, for example, in EP 331376, lipaseP.stutzeridisclosed, for example, in BP 1372034, lipaseP. fluorescensthe lipaseBacillusfor example, the lipaseB.subtilis(Dartois et al. (1993), Biochemica et Biophysica acta 1131, 253-260), a lipaseB.stearothermophilus(JP 64/744992), lipaseB.pumilus(WO 91/16422), lipasePenicillium camenbertii(Yamaguchi et al. (1991), Gene 103, 61-67), the lipaseGeotrichum candidum(Shimada, Y. et al. (1989), J. Biochem. 106, 383-388), and a variety of lipases fromRhizopussuch as lipaseR.delemar(Hass, M.J. et al. (1991), Gene 109, 117-113), lipaseR.niveus(Kugimiya et al. (1992), Biosci. Biotech. Biochem. 56, 716-719) and lipaseR.oryzae. Additional examples are cutinase fromPseudomonas mendocina(WO 88/09367), catenatus Fusarium solani pisi(WO 90/09446) and cutinase fromHumicola insolens(WO 2001/092502). The lipolytic enzyme may be a variant of the lipase described, for example, in WO 2000/060063.

Examples of commercially available lipases include LipexTM, LipoprimeTM, LipopanTM, Lipopan FTM, Lipopan XtraTM, LipolaseTM, LipolaseTMUltra, LipozymeTM, PalataseTM, ResinaseTM, NovozymTM435 and LecitaseTM(all available from Novozymes A/S). Other commercially available lipases include LumafastTM(lipasePseudomonas mendocinafrom Genencor International Inc.); LipomaxTM(lipasePs. pseudoalcaligenesfrom Gist-Brocades/Genencor Int. Inc.); and lipaseBacillussp. from Solvay enzyme. Additional lipase is available from other sources, such as lipase P "Amano" (Amano Pharmaceutical Co. Ltd.).

Suitable mannanase include mannanase bacterial or fungal origin. This includes chemically or genetically modified mutants. Examples of commercially available mannanase include MannawayTM(product of Novozymes) and MannaStar (product of Genencor).

Suitable LiAZ include LiAZ bacterial or fungal origin. This includes chemically or genetically modified mutants. Examples LiAZ include pectin-liasu and pectin-liasu. Examples of commercially available LiAZ are PectawashTMand PectawayTM(products Novozymes).

The present invention additionally pisanos by the following examples, which is not to be understood as limiting framework of the invention.

EXAMPLES

Example 1

A variety of peptide aldehydes were produced by a company specializing in peptide synthesis, all with a purity of >80%. Peptide aldehydes dissolved in DMSO to a concentration of 10 mg/ml prior to use.

Model liquid detergent get to test a variety of stabilizers:

The base detergent:

Component% wt./mass.
Alkylalkoxysilane sodium (C9-15, EU)6,0
Dodecylbenzenesulfonate sodium3,0
Toluensulfonate sodium3,0

Oleic acid2,0
Ethoxylates, primary alcohol (C12-15, EU)3,0
Ethoxylates, primary alcohol (C12-15, EU)2,5
Ethanol0,5
Monopropylene the ol 2,0
Trinatriytsitrat 2H2About4,0
Triethanolamine0,4
Deionized waterAdded 100%
the pH was brought to 8.5 with NaOH

Received reference detergent with enzymes:

Detergent A:

Component% wt./mass.
The base detergent99,0
Protease (Savinase 16,0 LEX)0,5
Lipase (Lipex 100L)0,5

Next I received the following detergents with the stabilizers according to the invention, all samples normalized to 100 g of detergent:

ID DetergentDetergent AndStabilizer (from a solution of 10 mg/ml)The molar excess of the inhibitor to the protease
B1/td> 100 g1.3 mg Z-RAY-H3
B2100 g2.2 mg Z-RAY-H5
B3100 g4.4 mg Z-RAY-H10
C1100 g2.8 mg Ac-GAY-H10
C2100 g7,0 mg Ac-GAY-H25
D1100 g1.8 mg Z-GAY-H5
D2100 g3.6 mg Z-GAY-H10
E1100 g1.4 mg Z-RVY-H3
E2100 g2,3 mg Z-RVY-H5
E3100 g4,6 mg Z-RVY-H10

F1100 g1.3 mg Z-LVY-H3
F2100 g2.1 mg Z-LVY-H5
F3100 g4.3 mg Z-LVY-H10
G1100 g1.1 mg Ac-LGAY-H3
G2100 g1.9 mg Ac-LGAY-H5
G3100 g3.7 mg Ac-LGAY-H10
H1100 g0.6 mg Ac-FGAY-H1,5
H2100 g1.2 mg Ac-FGAY-H3
H3100 g2 mg Ac-FGAY-H5
H4100 g4 mg Ac-FGAY-H10
H5100 g10 mg Ac-FGAY-H25
I1100 g1.2 mg Ac-YGAY-H3
I2100 g2.1 mg Ac-YGAY-H5
I3100 g4,2 mg Ac-YGAY-H10
J1100 g1.3 mg Ac-FGVY-H3
J2100 g2.1 mg Ac-FGVY-H5
J3100 g4.3 mg Ac-FGVY-H10
K1100 g1.5 mg Ac-WLVY-H3
K2100 g2.5 mg Ac-WLVY-H5
K3100 g5.1 mg Ac-WLVY-H10

Detergents put the sealed jars at 35°C and 40°C. Measured residual activity of lipase and protease (by comparison with a benchmark, which was stored at -18°C) at different points in time using a standard enzymatic analytical methods (protease activity was measured by hydrolysis of N,N-dimethylcathinone at 40°C, pH of 8.3, and the lipase activity was measured by hydrolysis of steam-nitrophenylacetate at 40°C, pH of 7.7). In the table below, 3x means 3-molar excess of inhibitor compared with protease etc.

DetergentResidual proteasa activity
1 week 40°With
The residual lipase activity
1 week 35°With
A (standard)11%3%
B1 (Z-RAY-H, 3)49%12%
B2 (Z-RAY-H, 5x)69%37%
B3 (Z-RAY-H, 10x)79%63%
C1 (Ac-GAY-H, 10x)59%
C2 (Ac-GAY-H, 25x) 73%62%
D1 (Z-GAY-H, 5x)55%22%
D2 (Z-GAY-H, 10x)77%49%
E1 (Z-RVY-H, 3)54%21%
E2 (Z-RVY-H, 5x)67%36%
E3 (Z-RVY-H, 10x)80%61%
F1 (Z-LVY-H, 3)32%7%
F2 (Z-LVY-H, 5x)43%15%
F3 (Z-LVY-H, 10x)59%33%
G1 (Ac-LGAY-H, 3)62%33%
G2 (Ac-LGAY-H, 5x)82%56%
G3 (Ac-LGAY-H, 10x)90%66%
H1 (Ac-FGAY-H, 1,5x)24%4%
H2 (Ac-FGAY-H, 3) 42%12%
H3 (Ac-FGAY-H, 5x)78%63%
H4 (Ac-FGAY-H, 10x)91%72%
H5 (Ac-FGAY-H, 25x)93%72%
I1 (Ac-YGAY-H, 3)53%14%
I2 (Ac-YGAY-H, 5x)90%66%
I3 (Ac-YGAY-H, 10x)88%75%
J1 (Ac-FGVY-H, 3)62%48%
J2 (Ac-FGVY-H, 5x)82%66%
J3 (Ac-FGVY-H, 10x)96%70%
K1 (Ac-WLVY-H, 3)26%3%
K2 (Ac-WLVY-H, 5x)35%8%

K3 (Ac-WLVY-H, 10x)53%18%

The results demonstrate that the tyrosine peptide aldehydes are very effective stabilizers proteases.

Example 2

Received reference detergent with enzymes:

Detergent L:

Component% wt./mass.
The basis of the detergent of example 198,5
Protease (Savinase 16,0 LEX)0,5
Lipase (Lipex 100L)0,5
Amylase (Stainton 12L)0,5

Received the following detergent with a stabilizer according to the invention and normalized to 100 g of detergent:

ID detergentDetergent LStabilizer (from a solution of 10 mg/ml)The molar excess of the inhibitor to the protease
M100 g2 mg Ac-FGAY-H5

Detergents were placed in Zack is itie glasses at 25°C and 35°C. Measured residual activity of lipase, amylase and protease (by comparison with a benchmark, which was stored at -18°C) at different time points (n=weeks) using standard enzymatic analytical methods (protease activity was measured by hydrolysis of N,N-dimethylcathinone at 40°C, pH of 8.3, and the lipase activity was measured by hydrolysis of steam-nitrophenylacetate at 40°C, pH of 7.7 and amylase activity was measured using hydrolysis 4,6-ethylidene-(G7)para-nitrophenyl-(G1)-α,D-maltoheptaoside at 37°C, pH 7,35).

% residual activityResidual proteasa activityThe residual lipase activityResidual amylase activity
Detergent4h 35°C13 h 25°C4h 35°C13 h 25°C4h 35°C13 h 25°C
L (standard)35%62%1%2%34%42%
M (Ac-FGAY-H, 5×)91%100%16%71%70%88%

It is seen that the tyrosine peptide aldehyde significantly improves the stability of protease, lipase and amylase in liquid detergent during storage.

Example 3

Peptide aldehydes Z-GAF-H, Z-GAL-H and Z-GAY-H were obtained using peptide synthesis, all with a purity of >80%. Peptide aldehyde was dissolved in DMSO to a concentration of 10 mg/ml prior to use.

Received the following detergent N with enzymes:

Component% wt./mass.
Alkylalkoxysilane sodium (C9-15, EU)20,0
Toluensulfonate sodium3,0
Oleic acid4,0
Ethoxylates, primary alcohol (C12-15, EU)2,5
Ethoxylates, primary alcohol (C12-15, EU)2,0
Ethanol2,1
Sodium carbonate4,5
Trinatriytsitrat 2H2About5,0
Deionized waterAdded 99%
the pH was brought to 8.0 with NaOH
Protease (Savinase 16.0 LEX)0,5
Lipase (Lipex 100L)0,5

Received the following detergents with a stabilizer according to the invention and normalized to 100 g of detergent:

Q3
ID detergentDetergent AndStabilizer (from a solution of 10 mg/ml)The molar excess of the inhibitor to the protease
P (ethanol)100 gNo0
Q1100 g0.16 mg Z-GAL-H0,5
Q2100 g0,31 mg Z-GAL-H1,0
100 g0,62 mg Z-GAL-H2,0
Q4100 g1.6 mg Z-GAL-H5,0
R1100 g0.17 mg Z-GAF-H0,5
R2100 g0,34 mg Z-GAF-H1,0
R3100 g0,68 mg Z-GAF-H2,0
R4100 g1.7 mg Z-GAF-H5,0
S1100 g0.18 mg Z-GAY-H0,5
S2100 g0.35 mg Z-GAY-H1,0
S3100 g0,71 mg Z-GAY-H2,0
S4100 g1.8 mg Z-GAY-H5,0

Detergents were placed is in closed beakers at 40°C. Measured residual protease activity (by comparison with a benchmark, which was stored at -18°C) after 1 week using standard enzymatic analytical methods (protease activity was measured by hydrolysis of N,N-dimethylcathinone at 40°C, pH 8,3).

% by residual activity after 1 week at 40°C:

The molar excess of the inhibitor to the proteaseDetergent N (standard)Detergent N + Z-GAL-HDetergent N + Z-GAF-HDetergent N + Z-GAY-H
07% (P)
0,512% (Q1)11%(R1)13%(S1)
117% (Q2)18%(R2)28%(S2)
231% (Q3)28%(R3)41%(S3)
42% (Q4)44%(R4)65%(S4)

The results demonstrate that all three peptide aldehyde are effective for the stabilization of the protease. It was found that the tyrosine peptide aldehyde Z-GAY-H is the most efficient because it requires only about half of the excess of the inhibitor to the protease to achieve the same residual activity, as with other peptide aldehydes.

1. Peptide aldehyde of formula B2-B1-B0-H, where
H means hydrogen;
In0represents the balance Tight;
B1represents one amino acid residue l or Val; and
B2represents one amino acid residue with attached benzyloxycarbonyl group (Cbz), or2represents two amino acid residue containing acetyl (AC) N-terminal protective group.

2. Peptide aldehyde according to claim 1, where In2represents one residue is Gly, Arg, or Leu with attached benzyloxycarbonyl (Cbz) group.

3. Peptide aldehyde according to claim 1, where In2consists of two amino acid residues, containing acetyl (AC) N-terminal protective group, where one of the residues In2represents Phe, Gly, or Leu, and the Torah In the balance 2represents Phe, Gly, Leu, Tight or Thr.

4. Peptide aldehyde of the formula Z-RAY-H, Ac-GAY-H, Z-GAY-H, Z-RVY-H, Z-LVY-H, Ac-LGAY-H, Ac-FGAY-H, Ac-YGAY-H, Ac-FGVY-H or Ac-WLVY-H, where Z is benzyloxycarbonyl, and AC represents acetyl.

5. Peptide aldehyde according to claim 4, which represents the Z-RAY-H, Z-GAY-H, Z-RVY-H or Z-LVY-H, where Z is benzyloxycarbonyl.

6. Liquid washing composition comprising surfactant, subtilisin and peptide aldehyde according to any one of claims 1 to 5.



 

Same patents:

FIELD: biotechnologies.

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EFFECT: invention allows removing protein contaminations from dishware with high efficiency and is safe for users and environment.

6 cl, 6 ex

FIELD: biotechnologies.

SUBSTANCE: invention proposes use of a composition containing transglycosidase ferment (EC 2.4.1.24) for decomposition of polysaccharide of natural gum. The above polysaccharide of natural gum is a substrate for the above ferment of transglycosidase. A natural gum destruction method is described, which involves contact of transglycosidase ferment with polysaccharide of natural gum for destruction of the above polysaccharide of natural gum. Besides, a cleaning method is proposed, which involves contact of an object contaminated with polysaccharide of natural gum with cleaning composition including transglycosidase ferment; and maintenance of the above object and cleaning composition under conditions sufficient for effective destruction of polysaccharide of natural gum, and thus, cleaning of the above object.

EFFECT: invention allows decomposing natural gums by means of transglycosidase ferment.

19 cl, 7 dwg, 4 ex

FIELD: biotechnologies.

SUBSTANCE: compositions containing active versions of alpha-amylase are proposed. Besides a new version of alpha-amylase, compositions as per the invention usually contain at least one additional ferment, a detergent, one surface-active substance, one complexing agent, an oxidiser, an acidifying agent, an alkaliser, a peroxide source, a harness source, salt, a detergent complexing agent, a polymer, a stabilising agent or a conditioner. Besides, application methods of those compositions for scouring of woven fabric, for washing or cleaning of products, such as dishware or linen, which are contaminated with starch-containing substances, are described.

EFFECT: improved thermal stability relative to parental; form of AmyS-like alpha-amylase, from which they have been obtained.

10 cl, 12 tbl, 24 dwg, 14 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a liquid hand dishwashing detergent composition which contains: (a) 0.001-10 wt % cationic polymer and (b) 0.005-3 wt % active inorganic mother-of-pearl agent having particle size smaller than 50 mcm, and the cationic polymer is a carboxyethyl cellulose salt. The present invention relates to a method of cleaning dishes.

EFFECT: obtaining a dishwasher detergent which cares for skin, particularly correction of cysts and skin lustre.

22 cl, 12 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of removing a biofilm from a surface based on treatment with perhydrolase and a mixture of enzymes, as well as the corresponding set and composition.

EFFECT: invention can be used to control biofilms in industry, dentistry and health care.

19 cl, 10 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: invention relates to hand dishwashing using a liquid detergent composition, which includes a step of applying said composition onto said dishes, said composition containing: (a) 6-32 wt % anionic surfactant, containing a sulphate surfactant in amount of not more than about 10% of the mass of the entire composition; (b) 0.005-3 wt % active mother-of-pearl agent; (c) 0.01-1 wt % rheology modifier; and (d) 0.01-5 wt % cationic polymer, wherein the rheology modifier contains micro-fibre cellulse.

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26 cl, 12 ex

FIELD: biotechnologies.

SUBSTANCE: peroxy hydrolase ferment is described, which may hydrolyze n-nitrophenylcaproate (pNC6) or n-nitrophenyloctanoate (pNC8) in presence of peroxide, where the specified ferment includes one of the following combinations of amino acid substitutes: Ala in the position 154 and Met in the position 194; Gly in the position 154 and Val in the position 194; or Gly in the position 12 and Met in the position 194, where the specified positions of amino acids are positionally equivalent to positions 12, 154 and 194 in the sequence SEQ ID NO: 2 of peroxy hydrolase M. smegmatis, and where the specified ferment produces peroxy acid. The following components are disclosed: produced nucleic acid, which codes the specified peroxy hydrolase ferment; recombinant nucleic acid, which expresses the peroxy hydrolase ferment, including a promotor and the specified produced nucleic acid; a vector of expression, including the specified recombinant nucleic acid; a host cell containing the recombinant nucleic acid and producing the peroxy hydrolase ferment. Compositions are described for washing, bleaching and disinfection, including effective amount of peroxy hydrolase ferment and source of hydrogen peroxide. Methods of washing, bleaching and disinfection are proposed, which include maintenance of a substrate in presence of the specified compositions for washing, bleaching or disinfection of the specified substrate, accordingly. Besides, it is suggested to use the specified produced peroxy hydrolase ferment for hydrolysis of an acyl ether substrate containing up to 8 atoms of carbon, in presence of hydrogen peroxide.

EFFECT: invention makes it possible to perform a reaction of hydrolysis of the specified compound in presence of hydrogen peroxide with formation of peroxy acids.

19 cl, 5 tbl, 4 ex

FIELD: biotechnologies.

SUBSTANCE: composition of neutral metalloprotease stabilised with an inhibitor is proposed to produce a liquid detergent solution. The composition contains from approximately 0.001% to approximately 10% wt of neutral metalloprotease and a competitive inhibitor. Besides, the competitive inhibitor represents a protein hydrolysate and is connected with at least approximately 90% of molecules of the specified neutral metalloprotease. Also the method is proposed to produce a composition of neutral metalloprotease stabilised with an inhibitor. The mixture is incubated, which contains at least one neutral metalloprotease and a protein substrate, in the water buffer at pH in the range from approximately 6.5 to approximately 11 and at the temperature from approximately 22°C to approximately 37°C. In process of incubation the substrate protein is split when exposed to metalloprotease, which results in formation of the hydrolysis product. The hydrolysis product is extracted with molecular weight of less than approximately 5000 Da and combined with neutral metalloprotease.

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12 cl, 3 dwg, 5 tbl, 3 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a dry detergent for automatic dish washing, containing: a) 80-95% base which contains one or more sulphate, carbonate, citrate and silicate, wherein the carbonate is present in an amount less than 25% of the composition; b) 0.1-10% nonionic surfactant; c) 0.55-4% stain-reducing system which contains (i) polyacrylate and (ii) carboxymethyl inulin, in which the ratio of polyacrylate to carboxymethyl inulin ranges from 2:1 to 3:1; and d) 0.1-3% enzyme system which contains (i) less than 0.2% Esperase 6.0T and (ii) alkaline stable protease including a balance. The present invention also relates to a method of reducing formation of water marks on dishware.

EFFECT: providing good dish washing while reducing undesirable stains and films on the surface of the dishware.

10 cl, 4 tbl, 2 dwg

FIELD: biotechnologies.

SUBSTANCE: compositions are proposed for cleaning of a fabric or a surface from a contaminating substance containing at least one triglyceride (versions). Compositions contain acyltransferase and alcohol substrate for acyltransferase. At the same time acyltransferase and alcohol substrate are present in the amount efficient for production of a detectable ester after combination of the specified composition with an acyl donor, which is a component of a stain or a contamination of the cleaned fabric or the cleaned surface. In one version acyltransferase is SGNH-acyltransferase. Also the methods are proposed for cleaning of a fabric or a surface from a contaminating substance containing at least one triglyceride (versions). The methods include combination of acyltransferase, alcohol substrate for it and an object contaminated with a substance containing the acyl donor. At the same acyltransferase calalyzes transfer of the acyl group from the acyl donor onto the alcohol substrate to produce a fabric care agent. In one version acyltransferase is SGNH-acyltransferase, and the fabric care agent is ester.

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33 cl, 18 dwg, 9 tbl, 14 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to ligands in the form of synthetic peptide amides of a kappa-opiate receptor, and namely to agonists of the kappa-opiate receptor, which show a low inhibition degree of P450 CYP and a low degree of penetration into brain. According to the invention, synthetic peptide amide is described by the following formula:

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19 cl, 11 dwg, 53 ex

FIELD: biotechnologies.

SUBSTANCE: peptides consist of four amino-acid residues that are used for stimulation of collagen production with fibroblast.

EFFECT: invention allows effective stimulation of collagenoses in fibroblast cells.

11 cl, 3 dwg, 7 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to peptide derivatives and peptidomimetics as transglutaminase inhibitors, methods for their preparation, pharmaceutical compositions containing said compounds as well as use of said transglutaminase inhibitors in particular for treatmenting coeliac disease and transglutaminase dependent diseases.

EFFECT: high efficiency of using said compounds.

12 cl, 20 dwg, 2 tbl, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to synthetic peptide amides of formula I

, which are agonists of a kappa-opiate receptor and show a low inhibition degree P450 CYP and lower brain penetration degree. Besides, the invention refers to pharmaceutical compositions containing the above compounds.

EFFECT: possibility of using compounds for prophylaxis and curing of pain and inflammation, which are related to different diseases and states.

17 cl, 9 tbl, 8 dwg, 39 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to stable alpha-melatotropin (α-MSH) peptide analogues which have affinity for melanocortin 1 receptor (MC1R), to pharmaceutical preparations of α-MSH peptide analogues, as well as to methods for using these analogues for treating the MC1R-related diseases in medicine and veterinary science.

EFFECT: preparing the stable alpha-melatotropin peptide analogues.

16 cl, 26 dwg, 2 tbl, 23 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to organic chemistry, more specifically to new biologically active peptides which can be used in pharmacology and medicine to create new drug preparations that have cytoprotective activity.

EFFECT: new biologically active peptides are presented.

12 ex, 28 dwg, 2 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing a peptide, which is characterised by that it involves conversion of the -SH group of the peptide which contains an amino acid residue having an -SH group to an -OH group, said method comprising the following steps from (a) to (c): (a) reaction of the -SH group in the peptide with a methylating agent to convert the -SH group to an -SMe group; (b) reaction of the -SMe group formed at step (a) with a cyanating agent to obtain an intermediate reaction product in form of an ester; (c) converting the intermediate reaction product obtained at step (b) to a peptide which contains an amino acid residue having an -OH group in more basic conditions than conditions at step (b).

EFFECT: improved method.

20 cl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: in claim described are organic compounds of formula I where radicals are given in description, which are applicable for elimination, prevention or alleviation of one or more symptoms, associated with HCV disorders.

EFFECT: obtaining pharmaceutical composition which possesses inhibiting activity with respect to NS3-4 HCV serinprotease, including formula I compound and pharmaceutically acceptable carrier.

30 cl, 25 ex, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: in claim described are organic compounds of formula I where radicals are given in description, which are applicable for elimination, prevention or alleviation of one or more symptoms, associated with HCV disorders.

EFFECT: obtaining pharmaceutical composition which possesses inhibiting activity with respect to NS3-4 HCV serinprotease, including formula I compound and pharmaceutically acceptable carrier.

30 cl, 25 ex, 2 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: in claim described are organic compounds of formula I where radicals are given in description, which are applicable for elimination, prevention or alleviation of one or more symptoms, associated with HCV disorders.

EFFECT: obtaining pharmaceutical composition which possesses inhibiting activity with respect to NS3-4 HCV serinprotease, including formula I compound and pharmaceutically acceptable carrier.

30 cl, 25 ex, 2 tbl

FIELD: biotechnologies.

SUBSTANCE: invention refers to application of oligopeptide chosen from a group consisting of tripeptide of general formula X-Pro-Tyr (X-P-Y), in which X can be chosen from a group consisting of Ile (I), Val (V), Ala (A), Trp (W), Leu (L), Phe (F), Gly (G), Glu (E) and Asn (N), or peptides including the above tripeptide, in medicine for immunomodulation. Besides, application of the above oligopeptide for production of remedy to be used as an immunomodulator is disclosed. Immunomodulating pharmaceutical composition containing an effective amount of the above oligopeptide, as well as a vaccine containing it are disclosed. Besides, a method for reinforcement of mammal immune system, as well as a method for improvement of immunogenic activity of vaccine composition using the above immunomodulating composition are disclosed. A method providing immunoreactivity to peptide or improvement of peptide immunoreactivity with a peptide compound with the above oligopeptide is disclosed.

EFFECT: use of oligopeptides in medicine for immunomodulation.

19 cl, 2 dwg, 2 tbl, 2 ex

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