WO1991016424A1 - Procede de modification appropriee de proprietes d'enzymes par transformation chimique et enzymes chimiquement transformes - Google Patents

Procede de modification appropriee de proprietes d'enzymes par transformation chimique et enzymes chimiquement transformes Download PDF

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Publication number
WO1991016424A1
WO1991016424A1 PCT/EP1991/000705 EP9100705W WO9116424A1 WO 1991016424 A1 WO1991016424 A1 WO 1991016424A1 EP 9100705 W EP9100705 W EP 9100705W WO 9116424 A1 WO9116424 A1 WO 9116424A1
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enzyme
formula
water
radical
enzymes
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PCT/EP1991/000705
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German (de)
English (en)
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Hans Ulrich Geyer
Gerhard Konieczny-Janda
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Hans Ulrich Geyer
Konieczny Janda Gerhard
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Publication of WO1991016424A1 publication Critical patent/WO1991016424A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2422—Alpha-amylase (3.2.1.1.) from plant source
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates

Definitions

  • the invention relates to a method for deliberately changing the properties of enzymes by chemically modifying them, to the production of chemically modified enzymes and to the chemically modified enzymes and their use.
  • German Offenlegungsschrift 29 19 622 discloses water-soluble stabilized enzyme derivatives, in particular protease derivatives, which, by reacting the enzyme with polysaccharide derivatives containing aldehyde groups, for example dialdehyde starch or dialdehyde cellulose, form an immobilized, ie insoluble Enzyme product and subsequent reduction of the immobilized enzyme product with complex hydrides were obtained.
  • polysaccharide derivatives containing aldehyde groups for example dialdehyde starch or dialdehyde cellulose
  • an immobilized, ie insoluble Enzyme product and subsequent reduction of the immobilized enzyme product with complex hydrides were obtained.
  • at least two of the available amino groups of the enzyme portion and at least two of the available aldehyde groups of the polysaccharide portion have to be crosslinked and the product obtained in this way has to be additionally hydrogenated.
  • subtilisin has already been nitrated, carbamylated, glutarylated and succinylated in the prior art in order to investigate the effects of these chemical modifications on the enzymatic activity.
  • ⁇ -Amylases have also already been treated with organic acid anhydrides - e.g. Acetic acid, propionic acid, butyric anhydride etc. - modified to the corresponding acyl-derivatized ⁇ -amylase.
  • Chymotrypsinogen was also treated with acid anhydrides such as e.g. Acetic or succinic anhydride, also converted into acylated derivatives and, for example, with ethylenediamine, into amidated derivatives.
  • the reaction of glucose isomerase or D-xylose isomerase with diethyl pyrocarbonate and of glucose isomerase with 2,4,6-trinitrobenzenesulfonic acid is also known.
  • the methods known in the prior art for the chemical modification of enzymes are only limited in their applicability to special enzymes and types of chemical modification with respect to the residues which can be introduced into the enzyme.
  • the known methods allow only little scope in terms of the manner and in terms of a graduated feasibility of the chemical modification of enzymes.
  • the reagents used in the prior art to modify the enzymes are often too aggressive or the functional groups (such as aldehyde groups or acid anhydride groups) contained in these reagents which are used to bind the modification reagents to the enzyme are too chemically reactive and make it difficult to control the Degree of modification. Or they are badly compatible with the enzymes themselves.
  • the chemical modification of enzymes in the prior art is therefore difficult to control according to the type and degree of modification.
  • the types of bonds formed between the enzyme and the modification reagent can also prove to be insufficiently stable to hydrolysis (such as, for example, imine, ester or amide bonds) and thereby restrict the use of the modified enzymes.
  • the object was therefore to provide a simple, mild and at the same time versatile process for the targeted change in the properties of enzymes by derivatization and for the chemical modification of enzymes, which can be used for a large number of enzymes. This procedure should take into account.
  • the type and degree of modification of the enzymes can be light, controllable and thus enable a customized adaptation of enzymes for specific requirements.
  • Another task was to provide new, valuable enzyme derivatives optimized by chemical modification for various applications.
  • the invention relates to a method for specifically changing the properties of an enzyme by chemical modification, which is characterized in that the enzyme is reacted with a terminal epoxy or halohydrin compound which specifically changes the cationic, biochemical and application properties of the enzyme, Contains amphiphilic, anionic and / or lipophilic or hydrophobic organic radical, converted into a water-soluble or non-water-insoluble, chemically modified enzyme (enzyme derivative).
  • the modification reagents used according to the invention for the chemical modification of enzymes contain a terminal epoxy group as a reactive group or a halohydrin group as their precursor. This epoxy or halohydrin group is a mild, chemically reactive group that preferably reacts with free basic groups of the enzyme to be modified.
  • Such basic groups in the enzyme to be modified are preferably free, primary amino groups of polyfunctional amino acids which are contained in the polypeptide chain making up the enzyme and are accessible to the reagent due to their position on the enzyme surface.
  • These are in particular, for example, the ⁇ -amino group of the amino acid lysine or, if appropriate, also the amino function in the guanidinium group of the amino acid arginine.
  • the basicity or nucleophilicity of these amino groups is favorably matched to the reactivity of the epoxy or halohydrin compounds used and the degree of modification can therefore be easily controlled by adjusting the reaction parameters pH, temperature and reaction time.
  • the epoxides or halohydrins used according to the invention are cheap and can be industrially produced in large quantities and are therefore readily available. A variety of these epoxies are also commercially available.
  • the epoxides can, by their nature, carry a large number of residues which, through their respective specific properties, modify the properties of the enzyme derivatives obtained and thereby the targeted variation of the biochemical and application properties enable these enzyme derivatives.
  • the invention also provides a process for the production of water-soluble or non-water-insoluble, chemically modified enzymes (enzyme derivatives), which is characterized in that little is done by reaction taking place under basic conditions at least one of the free primary amino groups of an enzyme not located in the activity center with a) epoxides of the formula I /
  • R represents a cationic, amphiphilic, anionic or lipophilic or hydrophobic organic radical, or with b) halohydrins of the formula II
  • X represents halogen, preferably chlorine or bromine
  • R has the meaning given above, converts the enzyme into a water-soluble or non-water-insoluble, chemically modified enzyme (enzyme derivative), in which one or more of the free amino groups mentioned of the enzyme with a radical of the formula A
  • water-soluble or non-insoluble, chemically modified enzyme derivatives are obtained, the water solubility of which essentially corresponds to that of the underlying enzymes or, in contrast, is possibly reduced to the extent that they are not completely water-insoluble.
  • water-soluble or water-solubility here also include fine dispersions, such as, in particular, emulsions.
  • the reaction is usually carried out in solution, preferably in an aqueous solution.
  • the reaction solutions can optionally contain enzyme-compatible organic solvents, for example alcohols, diols etc., or enzyme stabilizers.
  • Solid enzyme starting materials are therefore dissolved in the desired concentration in water or in the water / solvent mixture. If one starts directly from enzyme solutions from a normal enzyme production, then can be used in the resulting concentration or after concentration to the desired concentration.
  • aqueous enzyme concentrates are used which have a dry substance content of 10 to 35% by weight.
  • reaction it may prove expedient to carry out the reaction in the presence of enzyme stabilizers and / or solubilizers.
  • the reaction is then preferably carried out in the presence of propanediol, which may be present in amounts of up to about
  • the process according to the invention for producing chemically modified enzymes is carried out under basic conditions. This means that the reaction is carried out at pH values above 7. In a preferred embodiment of the production process according to the invention, the reaction is carried out at pH values from 9 to 11.
  • the reaction can either be started at a high initial pH value, which gradually shifts to smaller alkaline pH values (down to a maximum of pH 7) in the course of the reaction, or by constant readjustment of the pH value to a constant alkaline pH -Value to be held.
  • the pH value can be adjusted by all inorganic bases that are compatible with enzymes, e.g. in particular alkali oxides or hydroxides.
  • Sodium hydroxide in particular in the form of an aqueous solution (sodium hydroxide solution), is preferably used for pH adjustment.
  • the temperature of the process is only limited by the temperature stability of the enzymes used.
  • the implementation is therefore carried out in appropriate embodiments of the manufacturing method according to the invention Temperatures up to a maximum of 35 ° C.
  • the reaction is preferably carried out at temperatures of up to a maximum of 30 ° C.
  • the reaction temperature is generally limited by ambient temperatures of approximately 25 ° C., since no or only inadequate conversions can be observed below these temperatures.
  • the reaction time can range from a few hours to a whole day. As a rule, in expedient embodiments of the manufacturing process according to the invention, reaction times become. from 1 to 8 hours.
  • the degree of modification of the enzyme derivatives according to the invention obtained can be set in a wide range from low to medium and medium to strong. Instead of longer reaction times, it may also be appropriate, depending on the enzyme used, to shorten the reaction time by using an excess of modification reagent, which is not only advantageous from a process engineering point of view, but also from an economic point of view (higher value of the enzymes; cheap and in large amount of readily available modification reagents).
  • a 5 to 10-fold excess of modification reagent (based on the free, substitutable amino groups contained in the enzyme to be modified) is generally used.
  • modification reagent based on the free, substitutable amino groups contained in the enzyme to be modified
  • the one-time addition at the beginning of the reaction leads to higher reaction rates and also to higher ones Degree of modification and is therefore particularly useful when short reaction times or overall higher degrees of modification are to be achieved.
  • the reaction mixture is generally cooled to 20 ° C. and adjusted to a pH of 7 with an aqueous solution of an enzyme-compatible inorganic and / or organic acid.
  • An aqueous solution of citric acid may be mentioned as an example.
  • the reaction mixtures obtained can either be used as such for further use or can be subjected to conventional measures in a manner which is customary per se, such as in the case of normal enzyme production / packaging.
  • the mixtures containing the enzyme derivative according to the invention can be admixed with enzyme stabilizers which are conventional per se, worked up to concentrates or in a conventional manner, e.g. by spray drying, converted into a solid product and also granulated.
  • the invention provides a chemically very mild and enzyme-friendly process for the targeted chemical change in the properties of an enzyme by means of chemical modification, which is outstandingly suitable for the production of chemically modified enzymes.
  • the invention obtained by the process
  • Enzyme derivatives can be chemically modified enzymes, the underlying enzymes of which can be chemically modified in their properties by substitution with various types of organic residues.
  • the organic radicals R introduced into the enzyme with the epoxide of the formula I or the halohydrin of the formula II can be cationic, amphiphilic, anionic or hydrophobic.
  • the isoelectric points, the pH optima, the surface properties (charge, hydrophobicity), the interfacial activity etc. can be influenced in a targeted manner.
  • epoxides of the formula I or halohydrins of the formula II are used for the chemical modification of the underlying enzyme, in which R is a cationic organic radical of the formula - (CH 2 ) n -N + R 1 R 2 R 3 , in which R 1 , R 2 and / or R ⁇ are a lower alkyl radical having 1 to 2 carbon atoms, preferably methyl, and n is an integer from 1 to 3.
  • radicals R are then, for example, trimethylammoniummethylene, trimethylammoniumethylene, trimethylammoniumtrimethylene groups, the trimethylammonium ethylene group being very particularly preferred.
  • An example of a modification reagent used in this variant is, for example, 2,3-epoxypropyltrimethylammonium chloride.
  • R 1 and / or R 2 is a lower alkyl radical having 1 to 2 carbon atoms, preferably methyl, R 4 is a straight-chain C10 bis
  • C18 alkyl radical and n is an integer from 1 to 3.
  • modification reagents used in this variant are, for example, 3-chloro-2-hydroxypropyldimethyllaurylammonium chloride and 3-chloro-2-hydroxypropyldimethylcoco (C12-C16) ammonium chloride.
  • epoxides of the formula I or halohydrins of the formula II are used for the chemical modification of the underlying enzyme, in which R represents an anionic organic radical of the formula
  • chemical modification reagents used in this variant of the production process according to the invention are, for example, 2-chloro-1-hydroxyethylalkylene sulfonates, preferably in the form of the sodium salt.
  • Chemical modification reagents which are particularly preferred for this variant are, for example, the 3-chloro-2-hydroxypropylsulfonates, in particular that
  • Examples include ⁇ -epoxides such as hexen-1-oxide, octene-1-oxide, decen-1-oxide, dodecen-1-oxide, tetradecen-1-oxide, hexadecen-1-oxide and octadecen-1-oxide.
  • any enzymes per se in particular those with industrial importance for various applications such as detergent enzymes, enzymes for saccharification of starch and other applications can be used.
  • proteases, amylases are preferred embodiment of the production method according to the invention.
  • Glucose isomerases or lipases used.
  • other enzymes such as e.g. Pectinases, cellulases or hemicellulases etc. can be used.
  • the process according to the invention for the production of water-soluble, chemically modified enzyme derivatives allows targeted, chemically very mild tailoring of enzyme derivatives with properties which are adapted to the particular application in the desired manner.
  • the method according to the invention thus enables in a simple manner, e.g. to vary the surface properties with regard to charge and hydrophobicity in the desired manner.
  • Enzyme properties such as pH optimum, pH stability, temperature optimum, temperature stability, oxidation stability or compatibility with other enzymes (e.g. stability against digestion by protease, if present), interfacial activity etc. can be easily changed and adjusted to the respective technical application of the enzyme in the desired manner.
  • the invention therefore also relates to those water-soluble or non-water-insoluble, chemically modified enzymes (enzyme derivatives) which, in relation to their respective technical fields of application, are improved in their biochemical and application properties, these chemically modified enzymes being characterized in that they are present in at least one the free primary amino groups of the underlying enzyme which are not in the activity center and have a radical of the formula A
  • R is a cationic, amphiphilic, anionic or hydrophobic organic radical
  • Appropriate enzyme derivatives according to the invention are characterized in that the underlying enzyme is a protease, amylase, glucose isomerase or lipase.
  • the underlying enzyme can also be another, e.g. a pectinase, cellulase or hemicellulase etc.
  • the enzymes chemically modified according to the invention are distinguished by the fact that they are substituted by a radical of the formula A in which R represents a cationic radical of the formula - (CH 2 ) n -N + R 1 R 2 R 3 , wherein R 1 , R 2 and / or R 3 represent a lower alkyl radical having 1 to 2 carbon atoms, preferably methyl, and n is an integer from 1 to 3.
  • the underlying enzymes are proteases, as are used in particular for washing and cleaning compositions.
  • proteases protease derivatives
  • these alkaline or highly alkaline proteases can be obtained, for example, by cultivating Bacillus strains such as Bacillus subtilis, Bacillus alcalophilus, Bacillus amyloliquefaciens or Bacillus licheniformis etc.
  • enzyme derivatives which are distinguished by the fact that they are water-soluble or non-water-insoluble, chemically modified proteases which have a cationic on at least one of the free primary amino groups of the underlying protease which are not in the activity center organic radical of formula B.
  • R 1 , R 2 and / or R 3 are a lower alkyl radical having 1 to 2 carbon atoms, preferably a methyl radical, and n is an integer from 1 to 3, are substituted.
  • This cationic modification of proteases gives protease derivatives in which the positively charged ⁇ -amino groups of the lysine are quaternary
  • the enzyme derivatives according to the invention are distinguished by the fact that they are substituted by a radical of the formula A in which R represents an amphiphilic radical of the formula - (CH 2 ) n -N + R 1 R 2 R 4 , wherein R 1 and / or R 2 is a lower alkyl radical having 1 to 2 carbon atoms, preferably a methyl radical, R 4 is a straight-chain C10 to C18 alkyl radical and n is an integer from 1 to 3.
  • R represents an amphiphilic radical of the formula - (CH 2 ) n -N + R 1 R 2 R 4 , wherein R 1 and / or R 2 is a lower alkyl radical having 1 to 2 carbon atoms, preferably a methyl radical, R 4 is a straight-chain C10 to C18 alkyl radical and n is an integer from 1 to 3.
  • R 4 is a straight-chain C10 to C18 alkyl radical and n is an integer from 1 to 3.
  • the chemically modified enzymes are distinguished by the fact that they are substituted by a radical of the formula A in which R represents an anionic radical of the formula - (CH 2 ) n -SO 3 -, in which n is a means integer from 1 to 3.
  • the underlying enzymes are proteases, in particular alkaline or highly alkaline proteases for washing and cleaning compositions, as have already been described in more detail above.
  • water-soluble or non-water-insoluble, chemically modified enzymes which are distinguished by the fact that they are chemically modified proteases, in particular chemically modified alkaline or highly alkaline proteases, which are present in at least one of those which are not in the activity center located free primary amino groups of the underlying protease with an anionic organic radical of the formula CC
  • n is an integer from 1 to 3, are substituted.
  • the underlying enzyme is an ⁇ -amylase.
  • the underlying ⁇ -amylases are in particular those as are used for washing and cleaning compositions or, on the other hand, for example also for starch saccharification.
  • Thermostable ⁇ -amylase as can be obtained, for example, from BacillUs licheniformis, is particularly preferred.
  • water-soluble or non-water-insoluble, chemically modified enzyme derivatives which are characterized in that they are chemically modified ⁇ -amylases, preferably chemically modified thermostable ⁇ -amylases, which have at least one of the free primary amino groups of the underlying amylase which are not in the center of activity and have an anionic radical of the formula CC
  • n is an integer from 1 to 3, are substituted.
  • the anionically modified enzymes described above are particularly suitable for low-pH detergent systems such as heavy-duty liquid detergents and certain mild detergents.
  • the enzyme derivatives from mild alkaline proteases are particularly advantageous here, especially for bleach-free and largely builder-free liquid detergents.
  • the anionically modified enzymes have an increased stability compared to conventional liquid detergent components such as linear alkyl sulfonates, soaps, paraffin sulfonates, fatty acid ethoxysulfates etc. and are also advantageous in concentrated liquid detergents, especially those with a high water content and higher pH values Have storage stability.
  • the pH optimum of the anionically modified enzymes is generally shifted towards the acidic range by 0.5 to 1 pH unit compared to the underlying unmodified enzyme.
  • Another effect of the anionic modification is the greatly improved turbidity stability in liquid detergent formulations (ie the tendency of the enzyme dissolved in the liquid detergent or other constituents of the enzyme preparation dissolved therein to be stored flocculation is reduced).
  • the anionically modified enzymes described above, in particular also anionic ⁇ -amylases are also suitable for use in environmentally friendly enzyme-containing dishwashing detergents.
  • anionically modified ⁇ -amylase derivatives in the low pH range from 5.0 to 6.0 at higher temperatures (70 ° C.) have increased stability compared to the underlying unmodified ⁇ -amylases.
  • Another variant of the chemically modified enzymes according to the invention is characterized in that the underlying enzyme is substituted with a radical of the formula A in which R is a whole number for a hydrophobic organic radical of the formula - (CH 2 ) n -CH 3 where n from 1 to 17 means are substituted.
  • a radical of the formula A in which R is a whole number for a hydrophobic organic radical of the formula - (CH 2 ) n -CH 3 where n from 1 to 17 means are substituted.
  • modified enzymes with a hydrophobic character are modified enzymes with a hydrophobic character.
  • Protein (biuret) 15.1% by weight
  • the degree of modification here relates to the underlying protease (native protease), multiple substitutions not being taken into account, and is defined as the relative ratio of the concentration of the enzyme derivatives to the sum of the concentrations of the native enzyme and the enzyme derivatives.
  • the degree of modification is given in percent and a degree of modification of 100% is therefore synonymous with the absence of native enzyme.
  • the activity stated in% is volume-corrected and relates to the activity (delft units) of the native enzyme used.
  • the reaction was exothermic, causing the temperature set at the beginning to increase from 30 ° C. to approx. 33 to 35 ° C. After 4 hours, the reaction was stopped by cooling the reaction mixture to 20 ° C. (plate heat exchanger) and by adding aqueous 20% by weight citric acid with a pH of 7 being set.
  • the water-soluble, chemically modified protease was isolated by methods known per se for the isolation of enzymes, such as ion exchange chromatography.
  • the product obtained could be converted into enzyme granules without difficulty by measures known per se, such as extrusion or spray drying.
  • the results show that the active center of the chemically modified highly alkaline protease from Bacillus alcalophilus was not affected by the mild chemical modification process.
  • Example 1 Analogous to Example 1 was an aqueous enzyme concentrate of the following composition Enzyme: Alkaline protease from Bacillus licheniformis
  • Activity (DU / g): 921,000 adjusted to a pH of 10 with sodium hydroxide solution and mixed at ambient temperature (approx. 20 ° C.) with such an amount of a 70 to 71% by weight aqueous solution of 2,3-epoxypropyltrimethylammonium chloride, that amount of 5% by weight of active substance ( modification reagent) based on the amount of enzyme used.
  • modification reagent active substance
  • the reagent was added within 5 minutes.
  • the mixture was heated to 25 ° C. and the course of the reaction was followed by cation exchange chromatography as in Example 1.
  • the results of the course of the reaction are summarized below as a function of the reaction time.
  • the degree of modification is defined as in Example 1, the activity in percent (analogously to Example 1) is also volume-corrected and relates to the activity (delft units) of the native protease used.
  • reaction mixture was cooled and stopped by adding aqueous 20% by weight citric acid with a pH of 7.
  • the slight decrease in activity of the chemically modified protease compared to the native protease is due to a higher alkalinity of the alkaline Bacillus licheniformis protease used. Analogous modifications with other Bacillus licheniformis protease concentrates showed that this drop in activity can be avoided when using protanediol-containing protease concentrates. Propanediol-stabilized protease concentrates can be modified almost without loss.
  • the liquid end product of this example was used directly for further experiments.
  • an aqueous enzyme concentrate of the composition given in example 1 (highly alkaline protease from Bacillus alcalophilus) was reacted with 3-chloro-2-hydroxydimethylcoco (C12-C16) ammonium chloride and the course of the reaction was monitored analogously to example 1 by cation exchange chromatography.
  • the concentration of the protease was adjusted so that there was an activity of 200 kDU / ml in the reaction mixture.
  • the chlorine used as a chemical modification reagent hydrine was used in the form of the diluted, commercially available solution (Degussa) and used in an amount such that 2% by weight of active substance, based on the enzyme used, was present.
  • the reaction was carried out at 30 ° C. and the pH for activating and converting the chlorohydrin into the epoxide was adjusted constantly to pH 11 (trapping the hydrogen chloride released).
  • the degree of modification achieved as a function of the reaction time is summarized below.
  • aqueous enzyme concentrate of the highly alkaline protease from Bacillus alcalophilus with a propanediol content of 40% by weight 0.8 kg of aqueous enzyme concentrate of the highly alkaline protease from Bacillus alcalophilus with a propanediol content of 40% by weight and the following further composition
  • Protein (biuret) 6% by weight
  • Inorganic salts 3% by weight (e.g. CaCl 2 )
  • Non-symbol components 1% by weight
  • the isolation / concentration of the chemically modified proteases was carried out according to methods known per se for the isolation of enzymes, such as ion exchange chromatography.
  • alkaline proteases from Bacillus licheniformis were used in different ways.
  • the modified proteases obtained in this example have activities of over 90% of the starting activity (activity of the protease used).
  • Example 4 Analogously to Example 4, a Bacillus subtilis protease was reacted with sodium 3-chloro-2-hydroxyproylsulfonate under the following conditions. Enzyme: 38 g protease powder
  • aqueous enzyme concentrate made from highly alkaline protease from Bacillus alcalophilus was reacted with decen-1-oxide (Peroxid-Chemie) with intensive stirring and with the addition of a stabilizing and solvent-imparting amount of propanediol under the following conditions.
  • Reagent 2% by weight at the beginning of the
  • Pillar Waters Nova Pak C18.
  • Solvent A 0.1% by weight aqueous tri-fluoroacetic acid solution
  • Solvent B 0.1% by weight aqueous trifluoroacetic acid solution / acetonitrile in a volume ratio of 40/60
  • Example 2 Analogous to Example 2 was an aqueous standard amylase concentrate of the following composition
  • Enzyme Thermostable ⁇ -ylmylase from
  • Protein (biuret) 5% by weight
  • Inorganic salts 3% by weight (e.g. CaCl 2 )
  • the reaction could be stopped after 1 hour by cooling to 20 ° C. and adding aqueous 20% by weight citric acid with a pH of 7.
  • the anionically modified amylase derivative has a significantly increased stability compared to the native amylase at pH values in the range from 5.2 to 5.6.
  • glucose isomerase was chemically modified with 2,3-epoxypropyl-trimethylammonium chloride.
  • glucose isomerase crystals were dissolved in propanediol / water and reacted with the reagent (70 to 71% by weight aqueous solution of 2,3-epoxypropyltrimethylammonium chloride) under the following conditions.
  • Enzyme 160 g (38.4% by weight protein
  • glucose isomerase unit (* 1 glucose isomerase unit (GIU) is defined as the amount of enzyme that forms 1 mg fructose under the following incubation conditions; incubation conditions: 65 ° C, 1 h, substrate from 0.1 m glucose in 0.05 m phosphate buffer, pH 8, 0 with 0.0004 m MgSO 4. )
  • pH 5.8 column material based on diethylaminoethyl from Pharmacia LKB GmbH, Freiburg; Starting lumen 0.05 m NaH 2 PO 4 ; Eluens 0.05 m NaH 2 PO 4 / 0.8 m sodium chloride) are followed.
  • Example 12 Analogously to Example 10, pure glucose isomerase was reacted with sodium 3-chloro-2-hydroxypropylsulfonate, but the pH was kept constant at pH 11. No loss of activity for the chemically modified glucose isomerase compared to the native glucose isomerase was observed here either. This shows that the mild anionic modification for the native glucose isomerase is also very well tolerated. The liquid end product neutralized after termination of the reaction to pH 7 could be used directly for further experiments.
  • Example 12 Example 12:
  • the washing performance was determined by washing tests on test fabrics in laboratory washing machines (type: Linitest).
  • test fabric was washed with standardized detergent formulations which contained the chemically modified enzymes to be tested (enzyme derivatives) or, in comparative experiments, the native enzymes.
  • the tests were carried out at the specified pH values and with water at 15 ° dH in the temperature range from 15 to 60 ° C. specified in each case.
  • the washing performance was determined by measuring the lightening of soiled test fabrics as the remission difference (remission of the dry test fabric after the washing test minus the reflectance of the test fabric before the washing test). The bigger one
  • the washing tests were carried out as follows: The detergent solution containing enzymes worked in a rotating manner
  • magnesium silicate 2.0% by weight magnesium silicate, 25% by weight sodium perborate, 1.5% by weight tetraacetylethylene diamine and sodium sulfate ad
  • composition see under a) this
  • This cationically modified protease derivative degrades proteins such as egg proteins very well, d) conditions: 2 g / l heavy-duty liquid detergent, 30 ° C.,
  • European heavy duty liquid detergent containing 10.5% by weight of linear alkyl sulfonates, 13% by weight of sodium fatty acid salt, 5% by weight of alkyl sulfate, 10.3% by weight of fatty alcohol ethoxylate, 10% by weight of triethanolamine; 47% by weight of volatile components (water, propanediol).
  • the anionically modified enzyme derivatives were tested for their stability against individual liquid detergent constituents and against a complete liquid detergent formulation and for their storage stability.
  • LAS linear alkyl sulphonates
  • FES fatty acid ethoxysulphates
  • the anionically modified enzyme derivatives show significantly higher stabilities in the presence of the liquid detergent components compared to the native enzymes. While, for example, there is a rapid decrease in activity in the presence of anionic surfactants in the case of the native proteases, the moderately strongly anionically modified protease from Example 4b and the strongly anionically modified protease from Example 4c show considerably better stabilities than conventional anionic surfactants.
  • Anionically modified enzyme derivatives are very well suited for use in such liquid detergents due to their higher stability towards liquid detergent components. The results of the stability tests are summarized in the table below.
  • Triethanolamine 11.5% by weight
  • Liquid detergent formulation containing:
  • Enzyme derivatives This example shows the good stability of anionically modified proteases in concentrated liquid detergents of defined composition and with high water contents. Similarly good storage stabilities were also observed with the anionically modified protease from Example 6. Washing results with the stored samples confirmed the differences in storage stability found above through activity measurements. In contrast to the anionically modified proteases, after 50 days of storage the native enzymes had virtually no washing action.

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Abstract

Un procédé permet de modifier de manière appropriée les propriétés d'enzymes par transformation chimique. En outre, l'invention concerne de nouveaux enzymes chimiquement transformés, leur production et leur utilisation.
PCT/EP1991/000705 1990-04-25 1991-04-12 Procede de modification appropriee de proprietes d'enzymes par transformation chimique et enzymes chimiquement transformes WO1991016424A1 (fr)

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DEP4013142.4 1990-04-25
DE4013142A DE4013142C2 (de) 1990-04-25 1990-04-25 Verfahren zur gezielten Veränderung der Eigenschaften von Enzymen durch chemische Modifizierung und chemisch modifizierte Enzyme

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0579928A1 (fr) * 1992-05-25 1994-01-26 The Nisshin Oil Mills, Ltd. Lipase immobilisée, procédé de sa production et procédé de transestérification de huiles et graisses employant cette lipase
WO1995009909A1 (fr) * 1993-10-04 1995-04-13 Novo Nordisk A/S Preparation comportant une enzyme modifiee
WO1995026398A1 (fr) * 1994-03-28 1995-10-05 Novo Nordisk A/S Cellulase modifiee et preparation enzymatique la contenant
WO1998000500A1 (fr) * 1996-07-01 1998-01-08 Unilever Plc Composition detergente

Families Citing this family (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DZ3349A1 (fr) 2000-07-28 2002-02-07 Henkel Kgaa Nouvelle enzyme amylolytique issue de bacillus sp. a 7-7 (dsm 12368) ainsi que produits de lavage et nettoyage contenant ledit enzyme amylolytique
ES2290184T3 (es) 2000-11-28 2008-02-16 Henkel Kommanditgesellschaft Auf Aktien Ciclodextrina-glucanotransferasa (cgtasa) a partir de bacillus agaradherens (dsm 9948) asi como agentes para el lavado y la limpeiza con esta nueva ciclodextrina-glucanotransferasa.
DE10153792A1 (de) 2001-10-31 2003-05-22 Henkel Kgaa Neue Alkalische Protease-Varianten und Wasch- und Reinigungsmittel enthaltend diese neuen Alkalischen Protease-Varianten
DE10162728A1 (de) 2001-12-20 2003-07-10 Henkel Kgaa Neue Alkalische Protease aus Bacillus gibsonii (DSM 14393) und Wasch-und Reinigungsmittel enthaltend diese neue Alkalische Protease
DE10162727A1 (de) 2001-12-20 2003-07-10 Henkel Kgaa Neue Alkalische Protease aus Bacillus gibsonii (DSM 14391) und Wasch-und Reinigungsmittel enthaltend diese neue Alkalische Protease
DE10163748A1 (de) 2001-12-21 2003-07-17 Henkel Kgaa Neue Glykosylhydrolasen
DE10163884A1 (de) 2001-12-22 2003-07-10 Henkel Kgaa Neue Alkalische Protease aus Bacillus sp. (DSM 14392) und Wasch- und Reinigungsmittel enthaltend diese neue Alkalische Protease
DE10360805A1 (de) 2003-12-23 2005-07-28 Henkel Kgaa Neue Alkalische Protease und Wasch- und Reinigungsmittel, enthaltend diese neue Alkalische Protease
DE102004019751A1 (de) 2004-04-23 2005-11-17 Henkel Kgaa Neue Alkalische Proteasen und Wasch- und Reinigungsmittel, enthaltend diese neuen Alkalischen Proteasen
DE102007032111B4 (de) 2007-07-09 2017-07-20 Henkel Ag & Co. Kgaa Neue Proteasen und Wasch- und Reinigungsmittel enthaltend diese Proteasen
DE102007036756A1 (de) 2007-08-03 2009-02-05 Henkel Ag & Co. Kgaa Neue Proteasen und Wasch- und Reinigungsmittel, enthaltend diese neuen Proteasen

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2937012A1 (de) * 1978-09-18 1980-04-03 Henkel Kgaa Mittel zur stabilisierung von enzymen und deren verwendung
EP0210761A1 (fr) * 1985-07-05 1987-02-04 Takeda Chemical Industries, Ltd. Enzymes modifiées, leur production et utilisation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2937012A1 (de) * 1978-09-18 1980-04-03 Henkel Kgaa Mittel zur stabilisierung von enzymen und deren verwendung
EP0210761A1 (fr) * 1985-07-05 1987-02-04 Takeda Chemical Industries, Ltd. Enzymes modifiées, leur production et utilisation

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0579928A1 (fr) * 1992-05-25 1994-01-26 The Nisshin Oil Mills, Ltd. Lipase immobilisée, procédé de sa production et procédé de transestérification de huiles et graisses employant cette lipase
WO1995009909A1 (fr) * 1993-10-04 1995-04-13 Novo Nordisk A/S Preparation comportant une enzyme modifiee
US5866526A (en) * 1993-10-04 1999-02-02 Novo Nordisk A/S Enzyme preparation comprising a modified enzyme
WO1995026398A1 (fr) * 1994-03-28 1995-10-05 Novo Nordisk A/S Cellulase modifiee et preparation enzymatique la contenant
WO1998000500A1 (fr) * 1996-07-01 1998-01-08 Unilever Plc Composition detergente

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