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
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6478—Aspartic endopeptidases (3.4.23)
  • C12N9/6481—Pepsins (3.4.23.1; 3.4.23.2; 3.4.23.3)
• • 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
  • 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
  • C11D3/38672—Granulated or coated enzymes
• • 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
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
• • 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
  • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
  • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
• • 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
  • C12N9/14—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
  • C12N9/98—Preparation of granular or free-flowing enzyme compositions

Definitions

• the present invention relates to novel enzyme granules and a process for the preparation thereof.
• enzymes are produced by microorganisms such as bacteria, yeast and fungi. These enzymes are especially useful in detergents, starch and textile processing, and in feed and food applications. Examples of enzymes useful in detergent application include proteases, amylases, lipases and cellulases.
• the enzymes are produced by fermentation and then recovered, purified and concentrated into a liquid or brought in dry form. Suitable recovery techniques include filtration, centrifug- ation, membrane filtration, precipitation, crystallisation, chromatography, spray drying, etc.
• the dust levels in such preparations should be as low as possible.
• the dry enzymes are usually brought in granule form, for which several granulation techniques have been developed. See, for example, U.S. Patents Nos. 4,009,076, 4,016,040, 4,078,368, 4,242,219, etc.
• most of the granules are coated after their production with a film-forming macromolecular material.
• JP 58-179492A discloses the preparation of enzyme granules with a protective coating.
• a fluid bed dryer is used to first spray e.g. a liquid enzyme concentrate onto a core material and subsequently spray a coating material containing a cellulose derivative onto the granule, while drying takes place during the whole process.
• EP-A-0193829 describes a method for the production of dust free enzyme containing particles by coating hydratable core particles with an enzyme and then with a film-forming material. Coating is carried out by suspending the core particles in a fluidized bed dryer, spraying an aqueous slurry of enzyme onto the core particles while suspended, and evaporating water to leave a dried enzyme coat on the particles. The resultant enzyme-coated particles, while still suspended in the fluidized bed, are sprayed with a solution or dispersion of the macromolecular material, and dried to remove solvent to leave a coating of the macromolecular material.
• WO 90/09428 discloses a detergent additive granulate which comprises a core with a primary detergent additive, e.g. an enzyme, surrounded by a shell comprising a secondary detergent additive, e.g. another enzyme, a binder, and granulating agents, and optionally a filler, and a protective coating between the core and the shell, whereby the shell comprises cellulose or artificial fibres, and whereby the core optionally may also comprise cellulose or artificial fibres.
• the detergent additive granulate is said to exhibit a high physical strength, and the primary and secondary detergent additives are separated from each other and/or from harmful environmental factors.
• WO 93/07263 discloses a granular enzyme composition which comprises a core, an enzyme layer and an outer coating layer.
• the granular enzyme composition is said to have reduced tendencies to form dust and leave residue, and exhibit improved stability and delayed release characteristics.
• the method for making such enzyme- containing granules is said to have greatly reduced processing time.
• the present invention provides new enzyme granules with improved properties. As compared to the prior art cited above the granules according to the present invention have the advantage that the compound of interest is not only located at the surface of the granule.
• enzyme granules comprising porous core material in which the enzyme in solution is adsorbed.
• the enzyme-containing granules are preferably coated with a film-forming macromolecular material. Such granules have an improved mechanical strength.
• the invention further provides an efficient method of preparing the novel enzyme granules.
• the granules according to the present invention are based on core particles having a good pore size and pore size distribution to allow an enzyme solution to enter into the particle. Accordingly, the core material comprises the enzyme in liquid form, thus eliminating the drawback of processing powdered enzymes.
• the pore diameter should not be too big, since this will inter alia reduce the strength of the particle, and not too small, since this will prevent the compound of interest, e.g. an enzyme, entering into the pores.
• European Patent Application EP-A-0542351 (published on May 19, 1993, i.e. after the priority date of the present patent application) discloses a process for the preparation of salt granulates.
• the products which are obtainable by this process are very suitable for use as core material in enzyme applications.
• the level of porosity is important with respect to the economics of the process.
• a method for efficiently preparing the new enzyme granules which comprises the following steps: (a) preparing a non-aqueous enzyme solution
• the enzyme solution may be prepared in various ways, mainly depending on the physical characteristics of the enzyme used. Suitable solvents for the purpose of the invention include ethylene glycol, propylene glycol, liquid polyethylene glycols (PEG's) such as PEG 200 and PEG 400, and glycerol. In certain cases it may be useful to prepare first an aqueous enzyme solution and to add the non-aqueous solvent. The water is then partly or entirely removed, for example by evaporation.
• the "non-aqueous" enzyme solution may contain a certain amount of water, for example 10-20%, but this should not have an adverse affect on the various components of the granulate, in particular the porous core material.
• an enzyme slurry may be used in certain instances which will be clear to the man skilled in the art.
• the absorption of enzyme into the particle can be done in various way. Both from an enzyme containing aqueous liquid or slurry (e.g. a concentrated fermentation broth) as from an enzyme containing non-aqueous liquid or slurry (e.g. non ionics, alcohols etc.), or a combination thereof.
• Suitable porous core material includes soda, NaCl and silica and is commercially available.
• the porous core material is preferably prepared by the method described in EP-A-0542351.
• the enzyme solution can be brought onto the porous 5 core material in various ways which are all known in the art, for example using mixing devices or a fluidized bed or a mixer- fluid bed dryer combination. Process steps (b) and (c) may suitably be combined.
• one or more protective coating layer(s) o can be brought onto the core to yield a dust free enzyme containing particle.
• Suitable coating materials are frequently described in the literature, see e.g. JP 58-179492A and EP-A- 0193829. They include cellulose coatings or cellulose based coatings containing hydroxypropylcellulose, methyl cellulose, s hydroxypropyl methyl cellulose and/or hydroxyethyl cellulose. Also acrylic polymers like EUDRAGIT ® (Rohm Pharma) can be applied. The amounts of coating to be applied may vary in fairly wide ranges but is usually between 0.1 and 25% by weight, for the cellulose based coatings preferably between 5 o and 25 wt%.
• Coating layers can also be used to add other useful compounds to the granule. It is even possible to prepare multilayer granules wherein various coating layers have different functions, for example to stabilize the compounds 5 which are present, to add colour etc.
• porous core material which dissolves slowly or which liberates the absorbed enzyme slowly.
• This technology in combination with coating technology permits 0 a composition of a granule where several compounds can be released in a sequence according to demand.
• An example hereof is a granule where a protease is absorbed into a porous core, a lipase is coated on the core and finally a coating layer is brougth onto the granule. 5
• the entire process for preparing the enzyme granules according to the invention may be suitably carried out in one apparatus, for example a mixer or a fluidized bed.
• the process according to the invention has several advantages.
• the process is performed in more than one step the (enzyme) dust formation is reduced during transport of the particles between the apparatus.
• the absorption of an enzyme solution or slurry in a porous particle is much faster than e.g. spraying an enzyme solution or slurry onto a core in a fluid bed or than mixing an enzyme with a non-porous carrier.
• protease used in the examples is the high alkaline protease PB92, see U.S. Patent No. Re 30,602, which is commercially available from Gist-brocades N.V. under the trade mark MAXACAL ® .
• the lipase used in the examples is the lipase which is obtainable from the strain Pseudomonas pseudoalcaligenes M-l (CBS 473.85), see e.g. EP-B-0218272, and U.S. Patents Nos. 4,933,287 and 5,153,135.
• the chymosin used in the examples is produced by a transformed yeast strain of Kluyveromyces lactis and commercially available from Gist-brocades N.V. under the trade mark MAXIREN ® . See e.g. EP-B-0096430 and EP-B-0301669 and U.S. Patent No. 4,859,596.
• Soda cores with a porosity of 6 (Soda ash Dense ® ), 16 (Soda ash Compact ® ) and 38% (Soda ash Sorbent ® ), respectively, were obtained from Akzo N.V. (see also EP-A-0542351) .
• the soda was sieved to obtain a particle size between 315-710 ⁇ m.
• Spraying and absorption were carried out in a rotating vessel (enzyme liquid inlet temperature 30-50°C) .
• the material was then coated in a fluid bed dryer essentially according to the method described in JP 58-179492A or EP-A-0193829) and dust free enzyme granules were obtained.
• the non-aqueous protease solutions with ethylene glycol, propylene glycol, PEG 400 and glycerol were produced with a protease activity varying from 3.4 to 3.7 MADU/g.
• the liquid absorption process resulted in an enzyme yield of 61% to 98% based on enzyme activity, depending on the water content of the non-aqueous solution.
• the tests were carried out with soda cores of 6%, 16 and 38% porosity (see above).
• Protease granules were prepared in the same way as described in Example 1 but using porous NaCl cores instead of soda cores. These NaCl cores were prepared essentially according to the method described in EP-A-0542351 and had a porosity of 15%. These cores were filled with a non-aqueous liquid containing a protease with an activity of 3.4 MADU/g. This resulted in a granulate which had an activity of 440.000 ADU/g (enzyme yield >97%) . After coating in a fluidized bed with a cellulose based coating (20% w/w) under the same conditions as mentioned above, coated granules were obtained which had an activity of 397.500 ADU/g. Again a good mass yield was obtained (>92%) .
• Chymosin was dissolved in propylene glycol.
• the liquid absored well in the porous NaCl, which may absorb 15 % of its weight.
• Porous core material (Soda ash Compact ® , 600 g) with a particle size of 300-710 ⁇ m was introduced in a fluid bed dryer.
• the water was evaporated and the non-ionic and the lipase were absorbed into the particles.
• a coating layer was then sprayed onto the particles and dust-free granules were obtained.
• protease granules were made.
• the protease containing solutions of ethylene glycol and propylene glycol, respectively, were sprayed into the fluid bed directly on top of the porous material, which was fluidized (particle size between 400 and 600 ⁇ m) . Both when the protease was dissolved in ethylene glycol and propylene glycol, the liquid was absorbed well into the material.
• Enzyme dust [mg/20 g 0.45 0.31 0.14 0.20 of sample, based on
• porous core materials such as, NaCl and silica
• Ethylene glycol and polyethylene glycol were used as the solvents.
• Soda ash Sorbent ® (see Example 1) is able to absorb a liquid upto 37% by weight (based on the soda) , the NaCl used may absorb 15% of its weight, while the silica may absorb 100%.
• Table 2 shows the residual enzyme activity after absorption of various enzyme containing liquids into various core materials.
• the obtained material was tested for its protease stability at 7°C (at ambient relative humidity) using various non-aqueous liquids.
• Table 3 shows the results of the stability tests of the protease which is absorbed into the soda.
• EG ethylene glycol
• PG propylene glycol
• PEG polyethylene glycol 400

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• Chemical Kinetics & Catalysis (AREA)
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• Enzymes And Modification Thereof (AREA)
• Detergent Compositions (AREA)
• Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
• Confectionery (AREA)
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• 1994
  • 1994-05-18 EP EP94918349A patent/EP0675952A1/de not_active Ceased
  • 1994-05-18 CN CN94190299A patent/CN1110059A/zh active Pending
  • 1994-05-18 KR KR1019950700195A patent/KR950702625A/ko not_active Ceased
  • 1994-05-18 FI FI950183A patent/FI950183A0/fi not_active IP Right Cessation
  • 1994-05-18 AU AU69705/94A patent/AU677342B2/en not_active Ceased
  • 1994-05-18 PL PL94307854A patent/PL307854A1/xx unknown
  • 1994-05-18 WO PCT/EP1994/001642 patent/WO1994026883A2/en not_active Ceased
  • 1994-05-18 CA CA002140530A patent/CA2140530A1/en not_active Abandoned

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