EP2115111B1 - Dérivés de benzophénone ou d'anilide d'acide benzoïque portant des groupes carboxyle servant de stabilisateurs d'enzymes - Google Patents

Dérivés de benzophénone ou d'anilide d'acide benzoïque portant des groupes carboxyle servant de stabilisateurs d'enzymes Download PDF

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EP2115111B1
EP2115111B1 EP07847764.3A EP07847764A EP2115111B1 EP 2115111 B1 EP2115111 B1 EP 2115111B1 EP 07847764 A EP07847764 A EP 07847764A EP 2115111 B1 EP2115111 B1 EP 2115111B1
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group
protease
amino
cooh
acid
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EP2115111A1 (fr
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Andreas Michels
Robin Ghosh
Cornelius Bessler
Daniela Lowis
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Henkel AG and Co KGaA
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Henkel AG and Co KGaA
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    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38663—Stabilised liquid enzyme compositions

Definitions

  • the present invention relates to detergents containing carboxyl-bearing benzophenone or benzoic acid anilide derivatives which act as protease inhibitors and are thus suitable enzyme stabilizers.
  • enzymes in detergents are well established in the art. They serve to extend the range of services of the funds concerned according to their specific activities. These include in particular hydrolytic enzymes such as proteases, amylases, lipases and cellulases. The first three hydrolyze proteins, starches and fats and thus contribute directly to Schmutzentfemung. Cellulases are used in particular because of their tissue effect.
  • hydrolytic enzymes such as proteases, amylases, lipases and cellulases.
  • the first three hydrolyze proteins, starches and fats and thus contribute directly to Schmutzentfemung.
  • Cellulases are used in particular because of their tissue effect.
  • Another group of detergent enzymes are oxidative enzymes, in particular oxidases, which, if appropriate, together with other components, preferably serve to bleach soiling or to produce the bleaching agents in situ.
  • enzymes which are subjected to constant optimization, further enzymes are constantly being made available for use in detergents, in order in particular to be able to optimally treat special soiling, such as, for example, pectinases, ⁇ -glucanases, mannanases or further hemicellulases for the hydrolysis of, in particular, special vegetable polymers.
  • special soiling such as, for example, pectinases, ⁇ -glucanases, mannanases or further hemicellulases for the hydrolysis of, in particular, special vegetable polymers.
  • proteases and in particular serine proteases, which include the subtilases. They cause the degradation of protein-containing stains on the items to be cleaned. However, they also hydrolyze themselves (autoproteolysis) and all other proteins contained in the agents concerned, i. especially other enzymes. This happens especially during the cleaning process, i. in the aqueous wash liquor, if comparatively favorable reaction conditions are present. However, this also happens during the storage of the respective agent, which is why with increasing storage time always a certain loss of enzyme activities, such as the protease activity, accompanied.
  • the enzyme activity in the detergent is inversely proportional to the storage time, with increasing storage time the enzyme activity decreases more and more. This is particularly problematic in gel or liquid and in particular in water-containing formulations, because in this with the water contained both the reaction medium and the hydrolysis reagent are available.
  • One goal in the development of detergents is therefore to stabilize the enzymes contained, especially during storage.
  • the protection understood against various unfavorable influences such as against denaturation or decay by physical influences or oxidation.
  • One focus of these developments is the protection of the contained proteins and / or enzymes against proteolytic cleavage. This can be done by the construction of physical barriers, such as by encapsulation of the enzymes in special enzyme granules or by packaging the means in two- or multi-chamber systems.
  • Another frequently approached approach is to add chemical compounds to the agents which inhibit the proteases and thus act collectively as stabilizers for the proteases and the other proteins and enzymes contained. It must be reversible protease inhibitors, since the protease activity is only temporarily, especially during storage, but not be suppressed during the cleaning process.
  • Polyols in particular glycerol and 1,2-propylene glycol, benzamidine hydrochloride, borax, boric acids, boronic acids or their salts or esters are established as reversible protease inhibitors in the prior art.
  • These include, in particular, derivatives with aromatic groups, for example ortho, meta or para-substituted phenylboronic acids, in particular 4-formylphenylboronic acid (4-FPBA) or the salts or esters of the abovementioned compounds.
  • 4-FPBA 4-formylphenylboronic acid
  • peptide aldehydes that is oligopeptides with reduced C-terminus, especially those of 2 to 50 monomers, are described for this purpose.
  • peptidic reversible protease inhibitors include ovomucoid and leupeptin.
  • specific, reversible peptide inhibitors and fusion proteins from proteases and specific peptide inhibitors are used for this purpose.
  • polyols such as glycerol and 1,2-propylene glycol have proved to be unfavorable due to their high concentrations of use necessary, because the other active ingredients of the respective agents can thus be contained only in proportionally smaller proportions.
  • Boric acid derivatives occupy an outstanding position among the serine protease inhibitors, which are effective at comparatively low concentrations.
  • the international patent application discloses WO 96/21716 A1 that acting as protease inhibitors boric acid derivatives are also suitable to stabilize enzymes in detergents.
  • a selection of particularly powerful stabilizers are disclosed in the international patent application WO 96/41859 A1 ,
  • boric acid derivatives have a decisive disadvantage.
  • a protease is to be understood as meaning all enzymes which are capable of hydrolyzing acid amide linkages of proteins.
  • the proteases are also detailed below.
  • the compound represented by the general structural formula is an aromatic compound having two benzene rings linked by a keto or an acid amide group according to the feature (a). It is thus a benzophenone or benzoic acid anilide derivative.
  • This benzophenone derivative can according to the features (b) and (c) in both rings as radicals R1, R2, R3, R4 and R5 (in ring 1) or R6, R7, R8, R9 and R10 (in ring 2) Hydrogen (H), a carboxyl (COOH), a methyl (CH 3 ), an ethyl (C 2 H 5 ), a hydroxyl (OH), a hydroxymethyl (CH 2 OH), an amino (NH) 2 ) and / or carry a halogen.
  • the prerequisite is that in each of the two rings at least one carboxyl group (COOH) is present.
  • rings 1 and 2 can be distinguished, ring 1 being the one which can be attributed to the benzoic acid or its substitution product, and ring 2 being the one which can be attributed to the aniline or its substitution product.
  • a benzophenone or benzoic acid anilide derivative which has in one of the two rings 1 or 2 as possible substituents two of the radicals R 1 to R 10 (A) and (B) which are ortho-stable with one another ( A) a mandatory or optionally further carboxyl group (COOH) mentioned in (b) and / or (c) and (B) are a hydroxymethyl group present as such groups or optionally as a grouping -CH 2 -O-CO- and thus together represent a five-membered lactone with the C atoms of the ring carrying them.
  • a mandatory or optionally further carboxyl group (COOH) mentioned in (b) and / or (c) and (B) are a hydroxymethyl group present as such groups or optionally as a grouping -CH 2 -O-CO- and thus together represent a five-membered lactone with the C atoms of the ring carrying them.
  • two of the substituents (A) and (B) according to (b) and (c) are a carboxyl group (COOH) or a hydroxymethyl group, respectively, in a ring immediately adjacent, ie orthostatic to each other and are present in the form of the hydroxyl group and the carboxymethyl group side by side.
  • these two groups together form a lactone and bind in lactone form to the protease to be inhibited. It is also possible that the binding takes place without prior formation of the lactone form.
  • the realization of the present invention to predetermine the lactone form during the synthesis and to add the already formed lactone as stabilizer to the agent in question.
  • the most suitable form is to be determined experimentally on the basis of the protease to be inhibited and of the envisaged stabilizer, which does not pose any fundamental difficulties for the person skilled in the art.
  • this lactone carboxyl group is counted as a carboxyl group according to feature (b) and (c), respectively but may also be present in addition to another, carboxyl group.
  • the present invention encompasses the compounds mentioned in all protonated and / or deprotonated forms.
  • the carboxyl group (s) (COOH) and optionally the amino group (s) (NH 2 ) are present depending on the pH of the surrounding medium as carboxylate (COO - ) or as ammonium groups (NH 3 + ).
  • carboxylate COO -
  • ammonium groups NH 3 +
  • oppositely charged cations H + , Na + , K + or the like
  • anions Cl - , Br - , formate, acetate, etc.
  • the present invention can be realized. Decisive in each case is the interaction between the invention-relevant compound and the invention to be inhibited / stabilizing protease.
  • the compounds relevant to the invention form a complex with the protease to be inhibited / stabilized according to the invention.
  • the active site of the protease is blocked by a compound which is not hydrolyzable by this enzyme and is not available for hydrolysis of other proteins present.
  • the equilibrium coefficient of this reaction is called the inhibition constant or K i .
  • the first advantage of the compounds relevant to the invention over the prior art, in addition to their lower volume requirement compared with the polyols, is that they have favorable inhibition constants with respect to the proteases which can be used in detergents. This applies, for example, to serine proteases, but also to metalloproteases.
  • the inhibitors thus bind reversibly, i. they do not interfere with solid and not too loose transient interactions with the enzyme.
  • the majority of the protease relevant to the invention is thus present during storage in the form of a protease-inhibitor complex.
  • the protease and possibly other proteins contained, in particular other enzymes are protected in this way against proteolysis by this enzyme (stabilized against proteolysis).
  • the binding equilibrium is shifted in the direction of dissociation, so that the complex dissolves and most of the protease protease protease is proteolytically active.
  • the compounds relevant to the invention are functioning protease inhibitors and thus enzyme stabilizers for detergents in accordance with the task formulated.
  • the second advantage of the invention relevant compounds over the prior art is that they have as elements only C, H, N and O and optionally halides and / or sulfur and in particular are free of boron. They thus do not form the undesirable boron by-products with other detergent ingredients.
  • the compounds mentioned are presumed to act as reversible inhibitors because they bind the substrate of the proteases, in particular with regard to the acid amide bond to be hydrolyzed, structurally same.
  • all proteases can be inhibited by the compounds relevant to the invention, so that they are suitable as protease inhibitors according to the invention.
  • serine proteases as has been shown with reference to the examples of the present application with the positive effect of the compounds experimentally described there on the basis of serine proteases, specifically subtilases, even more specific subtilisins, namely a variant of the subtilisin from Bacillus lentus DSM 5483.
  • the stabilizing compound is selected from one of the following stabilizers: structural formula Surname a) 2- (4-carboxybenzoyl) benzoic acid b) 3,3'-carbonyl-benzoic acid c) 2- (3-carboxybenzoyl) benzoic acid d) 4,4'-carbonylbis-benzoic acid e) 2,2'-carbonyl-benzoic acid f) 3- (4-carboxybenzoyl) benzoic acid G) 2 - [[(3-carboxyphenyl) amino] carbonyl] -benzoic acid H) 2 - [[(4-carboxyphenyl) amino] carbonyl] -benzoic acid i) 2 - [(2-carboxybenzoyl) amino] benzoic acid j) 2-amino-2 ', 4-carbonylbis-benzoic acid k) 3 - [[(4-carboxyphenyl) amino] carbony
  • Such detergents are preferred according to the invention in which the stabilizing compound has an inhibition constant (Ki) of 0.01 to 10 mM, preferably 0.1 to 5, particularly preferably 0.5 to 2, with respect to the protease contained.
  • Ki inhibition constant
  • [E], [I] and [EI] represent the respective molar equilibrium concentrations of enzyme (E), inhibitor (I) and the enzyme-inhibitor complex (EI). According to this definition, a substance with a small Ki is a good inhibitor under the respective test conditions.
  • K i The determination of K i is carried out on the basis of the activity test of the protease in the presence of the corresponding inhibitor.
  • the kinetics of invertin action, Biochem. Z. 49: 333-369 ), the enzymatic parameters K m , and k cat are determined in the presence of various concentrations of the inhibitor. For a Michaelis-Menten kinetics is simplified:
  • K l can be calculated using the Cheng-Prusoff equation ( Equation 2, Cheng Y., Prusoff WH (1973) Biochem. Pharmacol. 22, 3099-3108 ) are determined via the IC 50 value.
  • the determination of the IC 50 via the determination of the catalytic activity to a substrate in the presence of various concentrations of the inhibitor and the fitting of the experimental data to a sigmoidal dose-response with variable slope equation (pseudo-Hill slopes). It is the inhibitor concentration needed to achieve 50% inhibition.
  • K i IC 50 / 1 + S / K d
  • [S] is the substrate concentration in the assay and K d is the dissociation constant for the substrate, which at the IC 50 concentration of the inhibitor can be considered to be identical to K m for the substrate.
  • a protease namely the Bacillus lentus alkaline protease F49 (according to WO 95/23221 A1 ) in the presence of an inhibitor. Since this is a typical subtilisin protease, the values obtained with this enzyme are also typical of other serine proteases, in particular other subtilisin proteases. The exact value for a protease of interest must be determined in doubt on the basis of each specific protease.
  • the protease is in particular in a content of 2 .mu.g to 20 mg per g of the agent, preferably 5 .mu.g to 17.5 mg per g of the agent, more preferably from 20 ⁇ g to 15 mg per g of the agent, most preferably from 50 ⁇ g to 10 ⁇ g of the agent.
  • the stabilizer is contained in agents according to the invention in particular in a content of up to 50 mg per g of the agent, preferably up to 10 mg, more preferably up to 7 mg, most preferably up to 5 mg per g of the agent. Furthermore, it is preferred that the stabilizer in a content of 0.01 to 100 x K i (based on the protease contained), preferably 0.1 to 10 x K l more preferably 1 to 5 x K i is included.
  • the molar ratio of stabilizer to protease is preferably in the range from 1: 1 to 1000: 1, in particular from 1: 1 to 500: 1, particularly preferably from 1: 1 to 100: 1, very particularly preferably from 1: 1 to 20 :1.
  • an agent according to the invention may contain at least one further stabilizer.
  • the detergent is thus characterized in that it contains at least one further stabilizer.
  • these compounds act synergistically, ie the stabilization effect achieved by both compounds exceeds the sum of the two individual stabilization effects.
  • the stabilizer (s) is one or more polyols, in particular glycerol or 1,2-ethylene glycol, an antioxidant, lactate or one or more lactate derivatives or combinations thereof. It is likewise preferably one or more of those enzyme-stabilizing or inhibiting compounds which are described in the international patent applications WO 07/113241 A1 or WO 02/008398 are disclosed.
  • the protease stabilized or reversibly inhibited according to the invention is preferably a serine protease, in particular a subtilase, more preferably a subtilisin.
  • subtilisins BPN 'and Carlsberg examples of such proteases are the subtilisins BPN 'and Carlsberg, the protease PB92, the subtilisins 147 and 309, the alkaline protease from Bacillus lentus, subtilisin DY and the enzymes thermitase, proteinase K which can no longer be assigned to the subtilisins in the narrower sense and the proteases TW3 and TW7.
  • Subtilisin Carlsberg is available in a further developed form under the trade name Alcalase® from Novozymes A / S, Bagsv ⁇ rd, Denmark.
  • the subtilisins 147 and 309 are sold under the trade names Esperase®, and Savinase® by the company Novozymes. From the protease from Bacillus lentus DSM 5483 derived under the name BLAP® protease variants derived.
  • proteases are, for example, under the trade names Durazym ®, relase ®, Everlase® ®, Nafizym, Natalase ®, Kannase® ® and Ovozymes ® from Novozymes, under the trade names Purafect ®, Purafect ® OxP and Properase.RTM ® from Genencor , under the trade name Protosol® ® from Advanced Biochemicals Ltd., Thane, India, under the trade name Wuxi ® from Wuxi Snyder Bioproducts Ltd., China, under the trade names Proleather® ® and protease P ® from Amano Pharmaceuticals Ltd., Nagoya, Japan, and the enzyme available under the name Proteinase K-16 from Kao Corp., Tokyo, Japan.
  • proteases are particularly well stabilized or reversibly inhibited by the compounds described.
  • certain variants of proteases i. also variants of said proteases, stabilized by these compounds particularly advantageous.
  • Such protease variants are part of the invention described below.
  • a protease stabilized or reversibly inhibited according to the invention can be a wild-type enzyme or a protease variant.
  • wild-type enzyme is to be understood that the enzyme is present in a naturally occurring organism or in a natural habitat can be isolated from this.
  • enzymes are modifiable and sometimes selectively modified, in particular in order to adapt their properties to the intended uses or to influence their catalytic activity. These changes often occur by altering the amino acid sequence of the enzyme. Such changes can be targeted and thus local or random, for example, by random mutagenesis done.
  • An enzyme variant is understood as meaning enzymes which have been generated from an initial enzyme, for example a wild-type enzyme, by altering the amino acid sequence.
  • the alteration of the amino acid sequence is preferably carried out by mutations, wherein amino acid substitutions, deletions, insertions or combinations thereof may be made.
  • the incorporation of such mutations into proteins is well known in the art and to those skilled in the art of enzyme technology. In principle, all enzymes can be changed in this way.
  • Protease variants are preferred according to the invention. These were generated from an initial protease, for example a wild-type protease, by altering the amino acid sequence, preferably amino acid substitutions, deletions, insertions or combinations thereof.
  • the starting protease does not necessarily have to be a naturally occurring wild-type protease; a protease known from the prior art in which changes have already been made can also be further developed and therefore again serve as an initial protease for generating further protease variants.
  • all of the proteases described above may be used unchanged in agents according to the invention and be stabilized by the compounds described.
  • they can also be the starting enzyme for a variant which is then contained in an agent according to the invention and stabilized by the compounds described.
  • the detergent is therefore characterized in that the protease has been obtained from an initial protease by at least one change of an amino acid, the change being a substitution, insertion or deletion of an amino acid, and at least to the starting protease at the amino acid level 90%, preferably at least 92.5%, more preferably at least 95%, and most preferably at least 97.5% is identical.
  • sequence comparisons are known to the person skilled in the art of enzyme technology.
  • sequence comparisons for example, the identity or homology values are determined for sequences to be compared.
  • Such a comparison is accomplished by associating similar sequences in the nucleotide or amino acid sequences of the proteins of interest. This is called homologization.
  • a tabular assignment of the respective positions is referred to as alignment.
  • alignments are created using computer programs, such as the algorithms FASTA or BLAST; This procedure is for example by DJ Lipman and WR Pearson (1985) in Science, Vol. 227, pp. 1435-1441 described.
  • a summary of all matching positions in the compared sequences is called a consensus sequence.
  • Such a comparison also allows a statement about the similarity or homology of the compared sequences to each other. This is represented in percent identity, that is the proportion of identical nucleotides or amino acid residues at the same or in an alignment corresponding positions. A broader concept of homology includes the conserved amino acid substitutions in this value. It then speaks of percent similarity. Such statements can be made about whole proteins or genes or only over individual areas.
  • homologous regions of different proteins are defined by matches in amino acid sequence. These can also be identified by identical function. It goes as far as complete identities in the smallest areas, so-called boxes, which contain only a few amino acids and usually perform essential functions for the overall activity.
  • the functions of the homologous regions are to be understood as the smallest partial functions of the function carried out by the entire protein, such as, for example, the formation of individual hydrogen bonds for the complexation of a substrate or transition complex.
  • sequence comparisons or alignments also serve to determine mutually corresponding positions in different molecules.
  • positions in the respective amino acid or nucleic acid sequence correspond to one another, even if the respective sequences have, for example, different total lengths or different domains or partial sequences or if additional amino acids or nucleotides are present within a sequence are.
  • a specific position in a first sequence can therefore be concretely assigned to a corresponding position in a second sequence, whereby it is quite possible for the positions corresponding to one another to be located at different locations in the molecule.
  • different amino acid residues may be present at the corresponding positions. Therefore, for such sequence comparisons or for determining a Specifically stated position, which position it is and which enzyme is assumed, that is, which counting method of determining position is to be based.
  • the amino acid sequence of the mature protein of the alkaline protease from Bacillus lentus DSM 5483 is used for determining the position, which is described in International Published Patent Application WO 91/02792 A1 and has a length of 269 amino acid residues (referred to in the present application as Bacillus lentus alkaline protease).
  • the detergent is characterized in that the protease was obtained from an initial protease by at least one change of an amino acid, the change being a substitution or insertion of an amino acid in that region of the amino acid sequence corresponding to positions 95 to 103 of the amino acid sequence Assigned alkaline protease from Bacillus lentus in an alignment.
  • Such a protease variant is particularly preferably a variant with an insertion of a single amino acid according to one or more of the positions 95, 96, 97, 98, 99, 100, 101, 102 and / or 103 and very particularly preferably between positions 97 and 98 and / or positions 99 and 100.
  • the detergent is characterized in that the protease has been obtained from an initial protease by at least one modification of an amino acid corresponding to the positions 3, 4, 36, 42, 43, 47, 56, 61, 69, 87, 96, 99, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 199, 205, 211, 224, 229, 236, 237, 242, 243, 250, 253, 255 and 268 of the Bacillus lentus alkaline protease are associated in an alignment, wherein the alteration is a substitution, insertion or deletion of an amino acid.
  • an amino acid change relative to the parent molecule occurs in one or more of the following positions: 3, 4, 43, 61, 188, 193, 199, 211, 224, 250 and 253 (count according to Bacillus lentus alkaline protease), more preferably with one or more of the amino acid substitutions X3T, X4I, X43V, X61A, X188P, X193M, X199I, X211L, X211D, X211E, X211G, X211N or X211Q, X224V, X250G and / or X253N.
  • the protease is a variant with a point mutation at position 211, preferably with a substitution of a single amino acid in this position, more preferably with the amino acid substitution X211 L.
  • the above positional information relates in turn to those amino acid residues which are assigned to said positions of the alkaline protease from Bacillus lentus in an alignment.
  • Agents according to the invention may contain, in addition to the protease, one or more further enzymes, in particular from the following group: one or more further proteases, amylases, hemicellulases, cellulases, lipases and oxidoreductases.
  • the amylase is preferably an ⁇ -amylase.
  • the hemicellulase is preferably a ⁇ -glucanase, a pectinase, a pullulanase and / or a mannanase.
  • the cellulase is preferably a cellulase mixture or a one-component cellulase, preferably or predominantly an endoglucanase and / or a cellobiohydrolase.
  • the oxidoreductase is preferably an oxidase, in particular a choline oxidase, or a perhydrolase.
  • Agents according to the invention preferably comprise at least one complexing agent and / or builder substances, the builder being in particular a zeolite builder, and / or a nonionic surfactant, the nonionic surfactant preferably being a hydroxy mixed ether, and / or optical Brightener, wherein the optical brightener is diphenyl compounds, in particular distyryl biphenyl derivatives, and / or stilbentriazine derivatives.
  • V1 is the strongest and thus most suitable protease inhibitor or stabilizer, followed by V2, V3, V4 (practically as good as V3) and V5.
  • these compounds are also suitable for stabilizing the enzymatic activities in protease-containing detergents and cleaning agents during storage.
  • a liquid detergent was prepared with the following composition (all figures in percent by weight): 0.3-0.5% xanthan gum, 0.2-0.4% anti-foaming agent, 6-7% glycerol, 0.3 -0.5% ethanol, 4-7% FAEOS, 24-28% nonionic surfactants, 1% boric acid, 1-2% sodium citrate (dihydrate), 2-4% soda, 14-16% coconut fatty acids, 0.5 % HEDP, 0-0.4% PVP, 0-0.05% optical brightener, 0-0.001% dye, balance: demineralized water.
  • the initial values for the proteolytic activity of the agent in question were compared with the values determined after storage. The higher the activity remaining after storage, the better the protease contained was inactivated during storage and the better the compound in question is suitable as a stabilizer according to the invention.

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Claims (15)

  1. Agent de lavage contenant une protéase et un composé répondant à la formule développée générale :
    Figure imgb0067
     dans laquelle
    (a) X représente un groupe carbonyle (C=O) ou un groupe d'amide d'acide (NHCO) ;
    (b) R1, R2, R3, R4 et R5 (dans le noyau 1) représentent un atome d'hydrogène (H), un groupe carboxyle (COOH), un groupe méthyle (CH3), un groupe éthyle (C2H5), un groupe hydroxyle (OH), un groupe hydroxyméthyle (CH2OH), un groupe amino (NH2) et/ou un atome d'halogène, au moins un groupe carboxyle (COOH) étant présent dans ce noyau ;
    (c) R6, R7, R8, R9 et R10 (dans le noyau 2) représentent un atome d'hydrogène (H), un groupe carboxyle (COOH), un groupe méthyle (CH3), un groupe éthyle (C2H5), un groupe hydroxyle (OH), un groupe hydroxyméthyle (CH2OH), un groupe amino (NH2) et/ou un atome d'halogène, au moins un groupe carboxyle (COOH) étant présent dans ce noyau ; et
    (d) de manière facultative, deux des résidus R1 à R10 (A) et (B), qui sont disposés en position ortho l'un par rapport à l'autre, (A) représentent un groupe carboxyle (COOH) obligatoire mentionné sous (b) et/ou (c) ou éventuellement un autre groupe carboxyle (COOH), et (B) un groupe hydroxyméthyle, qui sont présents sous la forme desdits groupes ou de manière facultative sous la forme d'un groupement -CH2-O-CO- et représentent ainsi ensemble avec les atomes de carbone du noyau qui les porte, une lactone à cinq membres.
  2. Agent de lavage selon la revendication 1, dans lequel le composé stabilisateur dans chacun des deux noyaux aromatiques porte de 1 à 3, de préférence 1 ou 2, de manière tout particulièrement préférée dans les deux noyaux ensemble 2 ou 3 groupes carboxyle, un groupe carboxyle intégré dans une lactone conformément à (d) étant compris dans le calcul.
  3. Agent de lavage selon la revendication 1 ou 2, dans lequel le composé stabilisateur présente, par rapport à la protéase qu'il contient, une constante d'inhibition (Ki) de 0,01 à 10 mM, de préférence de 0,1 à 5, de manière particulièrement préférée de 0,5 à 2.
  4. Agent de lavage selon l'une quelconque des revendications 1 à 3, dans lequel le composé stabilisateur est choisi parmi un des stabilisateurs indiqués ci-après : Formule développée Nom a)
    Figure imgb0068
         acide 2-(4-carboxybenzoyl)-benzoïque     b)
    Figure imgb0069
         acide 3,3'-carbonylbis-benzoïque     c)
    Figure imgb0070
         acide 2-(3-carboxybenzoyl)-benzoïque     d)
    Figure imgb0071
         acide 4,4'-carbonylbis-benzoïque     e)
    Figure imgb0072
         acide 2,2'-carbonylbis-benzoïque     f)
    Figure imgb0073
         acide 3-(4-carboxybenzoyl)-benzoïque     g)
    Figure imgb0074
         acide 2-[[(3-carboxyphényl)amino]carbonyl]-benzoïque     h)
    Figure imgb0075
         acide 2-[[(4-carboxyphényl)amino]carbonyl]-benzoïque     i)
    Figure imgb0076
         acide 2-[(2-carboxybenzoyl)amino]-benzoïque     j)
    Figure imgb0077
         acide 2-amino-2',4-carbonylbis-benzoïque     k)
    Figure imgb0078
         acide 3-[[(4-carboxyphényl)amino]carbonyl]-benzoïque     l)
    Figure imgb0079
         acide 4-[(4-carboxybenzoyl)amino]carbonyl]-benzoïque     m)
    Figure imgb0080
         acide 4-(2-carboxybenzoyl)-1,2-benzènedicarboxylique     n)
    Figure imgb0081
         acide 2-(2-carboxybenzoyl)-1,4-benzènedicarboxylique     o)
    Figure imgb0082
         acide 2-(4-carboxybenzoyl)-1,4-benzènedicarboxylique     p)
    Figure imgb0083
         acide 4-(3-carboxybenzoyl)-1,2-benzènedicarboxylique     r)
    Figure imgb0084
         acide 2-[[(1,3-dihydro-3-oxo-5-isobenzofuranyl)-amino]-carbonyl]-benzoïque     s)
    Figure imgb0085
         acide 2-[[(1,3-dihydro-1-oxo-5-isobenzofuranyl)-amino]carbonyl]-benzoïque
  5. Agent de lavage selon l'une quelconque des revendications 1 à 4, principalement sous forme solide.
  6. Agent de lavage selon l'une quelconque des revendications 1 à 4, principalement sous forme liquide, pâteuse ou sous forme de gel.
  7. Agent de lavage selon l'une quelconque des revendications 1 à 6, dans lequel la protéase est contenue en une teneur de 2 µg à 20 mg par g de l'agent, de préférence de 5 µg à 17,5 mg par g de l'agent, de manière particulièrement préférée de 20 µg à 15 mg par g de l'agent, de manière tout particulièrement préférée de 50 µg à 10 µg de l'agent.
  8. Agent de lavage selon l'une quelconque des revendications 1 à 7, dans lequel le stabilisateur est contenu en une teneur s'élevant jusqu'à 50 mg par g de l'agent, de préférence jusqu'à 10 mg, de manière particulièrement préférée jusqu'à 7 mg, de manière tout particulièrement préférée jusqu'à 5 mg par g de l'agent.
  9. Agent de lavage selon l'une quelconque des revendications 1 à 8, dans lequel le rapport molaire du stabilisateur à la protéase se situe dans la plage de 1:1 à 1000:1, en particulier de 1:1 à 500:1, de manière particulièrement préférée de 1:1 à 100:1, de manière tout particulièrement préférée de 1:1 à 20:1.
  10. Agent de lavage selon l'une quelconque des revendications 1 à 9, dans lequel le stabilisateur est contenu en une teneur de 0,01 à 100 x Ki (rapporté à la protéase contenue), de préférence de 0,1 à 10 x Ki, de manière particulièrement préférée de 1 à 5 x Ki.
  11. Agent de lavage selon l'une quelconque des revendications 1 à 10, dans lequel, en ce qui concerne la protéase, il s'agit d'une sérine-protéase de préférence d'une subtilase, de manière particulièrement préférée d'une subtilisine.
  12. Agent de lavage selon l'une quelconque des revendications 1 à 11, caractérisé en ce que
    (a) on obtient la protéase à partir d'une protéase de départ via au moins une modification d'un acide aminé, la modification représentant une substitution, une insertion ou une délétion d'un acide aminé, et elle est identique à la protéase de départ, sur le plan des acides aminés, à concurrence d'au moins 90 %, de préférence d'au moins 92,5 %, de manière particulièrement préférée d'au moins 95 % et de manière tout particulièrement préférée d'au moins 97,5 % ; et/ou
    (b) on obtient la protéase à partir d'une protéase de départ via au moins une modification d'un acide aminé, la modification représentant une substitution ou une insertion d' un acide aminé dans le domaine de la séquence d'acides aminés qui est attribué dans un alignement aux positions 95 à 103 de la protéase alcaline de Bacillus lentus, et/ou
    (c) on obtient la protéase à partir d'une protéase de départ via au moins une modification d'un acide aminé, qui est attribué dans un alignement aux positions 3, 4, 36, 42, 43, 47, 56, 61, 69, 87, 96, 99, 101, 102, 104, 114, 118, 120, 130, 139, 141, 142, 154, 157, 188, 193, 199, 205, 211, 224, 229, 236, 237, 242, 243, 250, 253, 255 et 268 de la protéase alcaline de Bacillus lentus, la modification représentant une substitution, une insertion ou une délétion d'un acide aminé.
  13. Utilisation d'un composé répondant à la formule développée générale
    Figure imgb0086
     dans laquelle
    (a) X représente un groupe carbonyle (C=O) ou un groupe d'amide d'acide (NHCO) ;
    (b) R1, R2, R3, R4 et R5 (dans le noyau 1) représentent un atome d'hydrogène (H), un groupe carboxyle (COOH), un groupe méthyle (CH3), un groupe éthyle (C2H5), un groupe hydroxyle (OH), un groupe hydroxyméthyle (CH2OH), un groupe amino (NH2) et/ou un atome d'halogène, au moins un groupe carboxyle (COOH) étant présent dans ce noyau ;
    (c) R6, R7, R8, R9 et R10 (dans le noyau 2) représentent un atome d'hydrogène (H), un groupe carboxyle (COOH), un groupe méthyle (CH3), un groupe éthyle (C2H5), un groupe hydroxyle (OH), un groupe hydroxyméthyle (CH2OH), un groupe amino (NH2) et/ou un atome d'halogène, au moins un groupe carboxyle (COOH) étant présent dans ce noyau ; et
    (d) de manière facultative, deux des résidus R1 à R10 (A) et (B), qui sont disposés en position ortho l'un par rapport à l'autre, (A) représentent un groupe carboxyle (COOH) obligatoire mentionné sous (b) et/ou (c) ou éventuellement un autre groupe carboxyle (COOH), et (B) un groupe hydroxyméthyle, qui sont présents sous la forme desdits groupes ou de manière facultative sous la forme d'un groupement -CH2-O-CO- et représentent ainsi ensemble avec les atomes de carbone du noyau qui les porte, une lactone à cinq membres,
    à titre d'inhibiteur réversible d'une protéase dans le cadre d'une formulation d'agent de lavage.
  14. Utilisation d'un agent de lavage selon l'une quelconque des revendications 1 à 12, pour le lavage et/ou le nettoyage des textiles.
  15. Utilisation d'une protéase et d'un composé répondant à la formule développée générale :
    Figure imgb0087
     dans laquelle
    (a) X représente un groupe carbonyle (C=O) ou un groupe d'amide d'acide (NHCO) ;
    (b) R1, R2, R3, R4 et R5 (dans le noyau 1) représentent un atome d'hydrogène (H), un groupe carboxyle (COOH), un groupe méthyle (CH3), un groupe éthyle (C2H5), un groupe hydroxyle (OH), un groupe hydroxyméthyle (CH2OH), un groupe amino (NH2) et/ou un atome d'halogène, au moins un groupe carboxyle (COOH) étant présent dans ce noyau ;
    (c) R6, R7, R8, R9 et R10 (dans le noyau 2) représentent un atome d'hydrogène (H), un groupe carboxyle (COOH), un groupe méthyle (CH3), un groupe éthyle (C2H5), un groupe hydroxyle (OH), un groupe hydroxyméthyle (CH2OH), un groupe amino (NH2) et/ou un atome d'halogène, au moins un groupe carboxyle (COOH) étant présent dans ce noyau ; et
    (d) de manière facultative, deux des résidus R1 à R10 (A) et (B), qui sont disposés en position ortho l'un par rapport à l'autre, (A) représentent un groupe carboxyle (COOH) obligatoire mentionné sous (b) et/ou (c) ou éventuellement un autre groupe carboxyle (COOH), et (B) un groupe hydroxyméthyle, qui sont présents sous la forme desdits groupes ou de manière facultative sous la forme d'un groupement -CH2-O-CO- et représentent ainsi ensemble avec les atomes de carbone du noyau qui les porte, une lactone à cinq membres,
    pour la production d'un agent de lavage.
EP07847764.3A 2007-03-06 2007-12-04 Dérivés de benzophénone ou d'anilide d'acide benzoïque portant des groupes carboxyle servant de stabilisateurs d'enzymes Not-in-force EP2115111B1 (fr)

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DE102007011236A DE102007011236A1 (de) 2007-03-06 2007-03-06 Carboxylgruppen tragende Benzophenon-oderBenzoesäureanilid-Derivate als Enzymstabilisatoren
PCT/EP2007/063260 WO2008107030A1 (fr) 2007-03-06 2007-12-04 Dérivés de benzophénone ou d'anilide d'acide benzoïque portant des groupes carboxyle servant de stabilisateurs d'enzymes

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PL2115111T3 (pl) 2014-09-30
EP2115111A1 (fr) 2009-11-11
ES2471447T3 (es) 2014-06-26
JP2010520336A (ja) 2010-06-10
WO2008107030A1 (fr) 2008-09-12
DE102007011236A1 (de) 2008-09-11
US7968508B2 (en) 2011-06-28

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