Classifications

• • 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
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
• • 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
  • C11D3/38654—Preparations containing enzymes, e.g. protease or amylase containing oxidase or reductase
• • 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
• • D—TEXTILES; PAPER
  • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
  • D06L—DRY-CLEANING, WASHING OR BLEACHING FIBRES, FILAMENTS, THREADS, YARNS, FABRICS, FEATHERS OR MADE-UP FIBROUS GOODS; BLEACHING LEATHER OR FURS
  • D06L4/00—Bleaching fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods; Bleaching leather or furs
  • D06L4/10—Bleaching fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods; Bleaching leather or furs using agents which develop oxygen
  • D06L4/12—Bleaching fibres, filaments, threads, yarns, fabrics, feathers or made-up fibrous goods; Bleaching leather or furs using agents which develop oxygen combined with specific additives

Definitions

• This invention relates to the stabilization of hemoproteins, particularly oxidoreductases such as peroxidase or catalase.
• oxidoreductases such as peroxidase or catalase are known to be hemoproteins. They can be obtained from many sources, e.g. from horseradish and microorganisms such as Coprinus (EP 179,486), Humicola or Scytalidium (WO 92/17571). Peroxidase can be added to a detergent together with a hydrogen peroxide precursor to improve the bleaching of stains (WO 89/09813) or to inhibit the transfer of dye from one textile to another (WO 91/05839). Other known uses of peroxidase are in the pulp and paper industry (EP 418,201 , EP 429,422, EP 433,258) and in production of particle boards (US 4,432,921).
• Catalase may be used for removing residual hydrogen peroxide after bleaching, e.g. of textiles. It is in some cases desirable to improve the stability of hemoproteins against denaturation and loss of enzyme activity, and it is the object of this invention to provide a method of stabilizing hemoproteins in solution.
• alkoxylated non-ionic compounds such as polyethylene glycol and alkylphenol ethoxylate, have a strong stabilizing effect on hemoproteins such as peroxidase in solution.
• US-A-4,414,326 and EP-A-0 272 578 describe the use of alkoxylated compounds of the above type in an enzymatic reagent composition for the determination of cholesterol.
• the compositions contained cholesterol esterase, cholesterol oxidase and peroxidase. Said documents describe activation and stabilization of cholesterol esterase and activation of cholesterol oxidase (US-A- 4,414,326 at col. 9 I. 26-28; EP-A-0 272 578 at col. 2 I. 18-20). No effect of the alkoxylated compound on peroxidase was described. Use of cholesterol oxidase and cholesterol esterase is not contemplated in the present invention.
• the invention provides a method of stabilizing a hemoprotein in an aqueous solution, characterized by incorporating a stabilizer of the formula:
• R-0-(CHR'-CH 2 -0-) n H (I) wherein R H or C ⁇ C ⁇ alkyl, aryl or alkylaryl
• the invention also provides a process comprising addition of peroxidase and hydrogen peroxide or a precursor thereof, characterized by incorporating said stabilizer.
• the invention also provides a peroxidase preparation for use in said bleaching composition or said process, in the form of a slurry, a non-dusting granulate or a stabilized liquid, characterized by comprising said stabilizer.
• the method of the invention may be applied to any hemoprotein, e.g. an oxidoreductase.
• hemoprotein e.g. an oxidoreductase.
• peroxidase EC 1.11.1.7
• Coprinus particularly C. cinereus, vide EP 179,486
• lignin peroxidase ligninase
• Phanerochaete chrysosporium or catalase e.g. from Humicola or Scytalidium
• haloperoxidases i.e. peroxidases that utilize halide ions and hydrogen peroxide to form carbon-halogen bonds in substrates, e.g.
• the hemoprotein may be produced by recombinant DNA technology (WO 92/16634).
• Other examples are cellobiose oxidase or cytochrome oxidase
• PODU Peroxidase Unit
• the invention provides increased activity, in addition to increased stability.
• the chain length (n) is preferably 50-500, particularly 100-300.
• the stabilizer may be alcohol ethoxylate or alkyl-phenol ethoxylate (propoxylate), where R is a C 10 -C 18 linear or branched alkyl or alkyl-phenyl group.
• the chain length (n) is preferably 5-50, particularly 5-20.
• the amount of said stabilizer should be such that the weight ratio of stabilizer to hemoprotein is 1-50.
• Suitable amounts of stabilizer are 0.1-5 g/l of aqueous solution, 1-30% by weight of bleaching composition or 1 -10% by weight of peroxidase preparation.
• a suitable ratio of stabilizer and peroxidase corresponds to 0.2-5 mg of stabilizer per PODU of peroxidase activity (unit defined below).
• the stabilizer is typically used at pH in the range 4 - 10, more preferably 6 - 8. Hydrogen peroxide source
• the source of hydrogen peroxide can be hydrogen peroxide itself, a precursor thereof or an enzymatic system capable of generating it.
• the precursor of hydrogen peroxide may be an inorganic or organic peracid or salt thereof, e.g. sodium perborate or sodium percarbonate.
• the enzymatic system may be a combination of a suitable oxidase and a substrate thereof, e.g. glucose and glucose oxidase.
• peroxidase or haloperoxidase may be used together with hydrogen peroxide or a precursor thereof e.g. in a process for bleaching of pulp for paper production (SE 88/0673, EP 418,201), in treatment of waste water from pulp production (US 4,623,465, JP-A 2-31887), in production of mechanical pulp from a fibrous product (EP 429,422, EP 433,258) and for lignin modification, e.g. in particle board production (US 4,432,921).
• Catalase may be used to remove hydrogen peroxide from fabric which is bleached by an alkaline hydrogen peroxide treatment before dyeing (GB 2,216,149; JP 2-104781).
• the peroxidase preparation of the invention may be provided as a slurry, a granulate or stabilized liquid, in a form suited for storage and shipping.
• Enzyme granulates may be made by known methods, e.g. by extrusion or drum granulation according to CA 974,907, US 4,106,991, US 4,435,307 or US 4,661 ,452.
• the stabilizer may be mixed with other ingredients in the core of the granules, preferably at above 1% by weight, preferably above 2%, particularly above 5%, and generally below 40%.
• the stabilizer may be incorporated by coating a preformed enzyme granulate. The amount of the stabilizer in the coating should make up at least 1% by weight of the coated particles.
• the coating may be applied by known methods e.g. as described in British Patent No. 1 ,362,365, page 1 , line 82 to page 2, line 34, and British Patent application No. 34973/73 and 10842/74 corresponding to Belgium patent No. 146,802.
• the coating material may comprise, in addition to the ethoxylated compound, other waxy coating materials and particulate material, e.g. magnesium silicate, alumino silicates, CaSO 4I CaCO 3 , bentonite, zeolite or TiO 2 .
• a liquid enzyme preparation according to the invention may be stabilized against microbial infection by incorporation of a stabilizing agent known in the art.
• a stabilizing agent known in the art.
• examples are inorganic salts (such as NaCI), sugars (such as sucrose and glucose), polyols (such as glycerol, propylene glycol and sorbitol) and alcohols
• organic acids such as benzoic, sorbic, propionic, lactic and formic
• stabilizing agents are antioxidants (such as sulphur dioxide), 1 ,2-benz-iso-thiazolin-3-one (BIT) and parabens.
• the amount of the dispersant in the liquid enzyme preparation is preferably 1-10% by weight.
• An enzyme slurry may be produced according to WO 91/09941.
• Peroxidase activity is determined by an assay based on the oxidation of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate) (ABTS ® ) by hydrogen peroxide (as described by Bergmeyer H.U., Methods of enzymatic analysis, 3rd edition, vol III, p. 286-293, 1983, modified). The greenish-blue colour produced is photometered at 418 nm.
• the analytical conditions are 0.88 mM hydrogen peroxide, 1.67 mM ABTS, 0.1 M phosphate buffer, pH 7.0, 30°C incubated for 3 minutes.
• 1 peroxidase unit is the amount of enzyme that catalyzes the conversion of 1 xmol hydrogen peroxide per minute at these conditions.
• the concentration of Triton X 405 in the peroxidase samples was varied from 0 to 1.5 g/l before incubation under the conditions described in Example 2, except that the temperature was 0°C.
• the enzyme activity was determined at 0, 1.5 and 3.5 hour.
• Table 3 It appears from Table 3 that an improved peroxidase stability is obtained by addition of 1 or 1.5 g/l of Triton X 405 to the 0.2 ml peroxidase sample (corresponding to a concentration of Triton X 405 of about 0.1 g/l during incubation).
• the concentration of Coprinus peroxidase in the samples was varied from 0.3 - 0.6 PODU/ml to 3 - 6 PODU/ml.
• the concentration of Triton X 405 in the peroxidase sample was 1.5 g/l.
• Incubation with ABTS ® was carried out as described in Example 2, except that the temperature was 22°C.
• the enzyme activity was determined at 0, 1 , 2, and 3 hours. The results are shown in Table 4.
• Triton X 405 stabilizes the peroxidase activity for up to 3 hours at both the lower and the higher peroxidase concentrations.
• a catalase solution was obtained from a culture broth filtrate prepared according to WO 92/17571 by adding 15% propylene glycol and adjusting to pH 7.0. Stabilizer was added as shown in Table 5. Each solution was stored at 25 or 37°C for 2 or 4 weeks, the catalase activity was determined before and after storage, and results were expressed as residual activity in % of the initial activity. The same stabilizers were used as in Example 1.

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• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Health & Medical Sciences (AREA)
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• Wood Science & Technology (AREA)
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• General Engineering & Computer Science (AREA)
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• Oil, Petroleum & Natural Gas (AREA)
• Enzymes And Modification Thereof (AREA)

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Citations (2)

• 1992
  • 1992-10-28 DK DK921317A patent/DK131792D0/da not_active Application Discontinuation
• 1993
  • 1993-10-28 WO PCT/DK1993/000345 patent/WO1994010298A1/en not_active Application Discontinuation
  • 1993-10-28 EP EP93924531A patent/EP0669975A1/en not_active Withdrawn
  • 1993-10-28 JP JP6510574A patent/JPH08502411A/ja not_active Withdrawn

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