WO2009030728A2 - Compositions enzymatiques contenant un constituant de stabilisation - Google Patents

Compositions enzymatiques contenant un constituant de stabilisation Download PDF

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Publication number
WO2009030728A2
WO2009030728A2 PCT/EP2008/061695 EP2008061695W WO2009030728A2 WO 2009030728 A2 WO2009030728 A2 WO 2009030728A2 EP 2008061695 W EP2008061695 W EP 2008061695W WO 2009030728 A2 WO2009030728 A2 WO 2009030728A2
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Prior art keywords
alpha
amylase
enzyme
composition
activity
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PCT/EP2008/061695
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English (en)
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WO2009030728A3 (fr
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Lone Aslaug Hansen
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Novozymes A/S
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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
    • C11D3/38609—Protease or amylase in solid compositions only
    • 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
    • C11D3/02—Inorganic compounds ; Elemental compounds
    • C11D3/04—Water-soluble compounds
    • C11D3/042—Acids
    • 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
    • C11D3/02—Inorganic compounds ; Elemental compounds
    • C11D3/04—Water-soluble compounds
    • C11D3/046—Salts
    • 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
    • C11D3/02—Inorganic compounds ; Elemental compounds
    • C11D3/04—Water-soluble compounds
    • C11D3/046—Salts
    • C11D3/048—Nitrates or nitrites
    • 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
    • C11D3/38663—Stabilised liquid enzyme compositions
    • 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.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C12N9/242—Fungal source
    • 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 present disclosure relates to alpha-amylase compositions having improved storage stability. More particularly, it relates to compositions where the alpha-amylase includes a carbohydrate-binding domain (CBD).
  • CBD carbohydrate-binding domain
  • the compositions are useful for enzyme compositions (e.g., industrial enzyme products) formulated to exhibit extended shelf life and/or decreased degradation of enzyme activity such as alpha-amylase activity.
  • the present disclosure further relates to processes for the manufacture of such stabilized compositions.
  • Alpha- Amylases (e.g., alpha-1 ,4-glucan-4-glucanohydrolases, EC. 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1,4- glucosidic oligo- and polysaccharides.
  • alpha-amylase referred to as "Termamyl®-like alpha-amylases" and variants thereof are known from, e.g., WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873 and WO 96/23874.
  • Termamyl-like alpha-amylases are suitable for processes carried out at high temperatures such as starch liquefaction in dextrose production processes.
  • US 20060148054 entitled Enzymes for starch processing discloses alpha- amylases which include a carbohydrate-binding domain, such as hybrids for starch processing.
  • WO200129167 discloses an aqueous detergent composition comprising a reducing alkali metal salt (e.g. sulfite) and an amylase.
  • WO200181617 discloses determination of amylase in the presence of sulfite.
  • stabilizing constituent such as reducing salts (sulfites etc.) have a significant effect on the storage stability of alpha-amylase, and that such enzymes can be efficiently formulated in a broadened pH range such as a pH of about 3.5 to about 11 by the addition of a stabilizing constituent. Accordingly, it is now possible to formulate enzyme compositions using a stabilizing constituent to provide enzyme(s) that retain at least 60%, 70%, 80% or 90% of its initial activity at ambient temperature or room temperature for at least about 13 weeks after forming the composition.
  • stabilizing constituent such as reducing salts (sulfites etc.) have a significant effect on the storage stability of alpha-amylase, and that such enzymes can be efficiently formulated in a broadened pH range such as a pH of about 3.5 to about 11 by the addition of a stabilizing constituent. Accordingly, it is now possible to formulate enzyme compositions using a stabilizing constituent to provide enzyme(s) that retain at least 60%, 70%, 80% or 90% of its initial activity at ambient temperature or room temperature for at
  • enzyme compositions using a stabilizing constituent to provide enzyme(s) that retain at least 60%, 70%, 80% or 90% of its initial alpha-amylase activity at ambient temperature or room temperature for at least about 13 weeks after forming the composition. Further, it is now possible to formulate enzyme compositions using a stabilizing constituent to provide enzyme(s) that retain more than 60%, 70%, 80% or 90% of its initial alpha-amylase activity at 40 degrees temperature for at least about 4 weeks after forming the composition. Some embodiments formulate alpha-amylase enzyme compositions with improved storage stability.
  • the invention provides a method of stabilizing an enzyme composition
  • an enzyme composition comprising stabilizing constituent in an amount of about 0.01% to about 20% by weight of the total composition, and in embodiments in an amount of about 0.5% to about 10%, and in embodiments in amount of about 0.1% to about 0.6% by weight of the total composition.
  • the stabilizing constituent may be added to enzyme having alpha-amylase activity after the fermentation, recovery and/or purification of the enzyme.
  • the invention also provides stable enzyme compositions which include one or more enzymes contacted with the stabilizing constituent of the present disclosure.
  • Such compositions may be formulated to maintain excellent stability in a broadened pH range (e.g., 3.5-11 or 4-7 or 4.5-5).
  • the invention further provides stable liquid enzyme compositions which include one or more enzymes contacted with the stabilizing constituent of the present disclosure.
  • Such liquid compositions may be formulated to maintain excellent stability in a broadened pH range (e.g., 3.5-11 or 4-7 or 4.5-5).
  • compositions exhibiting excellent alpha-amylase stability and including one or more stabilizing constituents as described herein.
  • Such compositions can be formulated into products with increased shelf life.
  • the excellent stability also leads to product forms such as, for example, solutions of enzymes having alpha-amylase activity manufactured and/or formulated at an increased and/or broadened pH.
  • the present compositions may further include a solvent constituent in which the enzyme and stabilizing composition are soluble.
  • the compositions have been found to be useful in forming liquid enzyme solutions including enzymes mixtures where some enzymes have alpha-amylase activity and other enzymes do not have alpha-amylase activity.
  • suitable stable enzyme compositions in accordance with the present disclosure provide a solution formulation suitable for use in a raw starch hydrolysis process that converts starch to sugar, and then ferments to ethanol with and/or without heat.
  • the enzyme formulations are provided in product forms such as liquids.
  • the objects of the present disclosure are met by providing an enzyme composition comprising one or more enzymes having alpha-amylase activity, and stabilizing constituent present in an effective amount to retain at least 60% of the enzymes initial activity at ambient temperature for at least about 13 weeks after forming the composition.
  • the objects of the present disclosure are met by providing a method of increasing the stability of enzyme having alpha-amylase activity comprising contacting a stabilizing constituent with one or more enzymes to provide a stable enzyme composition, wherein the enzyme retains at least 60% of its initial alpha-amylase activity at ambient temperature for at least about 13 weeks after forming the composition.
  • the objects of the present disclosure are met by providing an enzyme composition comprising at least two or more enzymes, and one or more stabilizing constituents in an amount of about 0.1 to about 20% by weight of the total composition, wherein at least one enzyme has an initial alpha-amylase activity, and wherein the enzyme composition is formulated to retain at least 60% of its initial acid alpha-amylase activity at ambient temperature for at least about 13 weeks after forming the composition.
  • ambient temperature means the nominal temperature of the air (or other gases, liquids, etc.) that surrounds the component, module, assembly, or system. As used herein the term further refers to a temperature in an amount of between O 0 C and 40 0 C.
  • room temperature refers to the temperature of the air (or other gases, liquids, etc.) in an amount of 23°C ⁇ 2 0 C.
  • sulfite refers to any salt of sulfurous acid, including but not limited to any compounds that contain sulfite ion (SO 3 2" ), bisulfite ion (HSO 3" ), meta bisulfite ion (S 2 O 5 2" ) and hydrosulfite ion (S 2 O 4 2" ) and/or combinations thereof.
  • alpha-amylase activity refers to hydrolase activity, e.g., hydrolytic EC. 3.2.1.1 , which catalyze hydrolysis of starch and other linear and branched 1,4- glucosidic oligo- and polysaccharides.
  • alpha-amylase activity may be expressed in, among other things, "FAU/F” as described further below.
  • variant refers to a polypeptide having alpha-amylase activity comprising alterations, such as substitutions, insertions, and/or deletions, of one or more (several) amino acid residues at one or more (several) specific positions of the mature polypeptide.
  • wild-type alpha-amylase denotes an alpha-amylase expressed by a naturally occurring microorganism, such as yeast or filamentous fungus found in nature.
  • mature polypeptide is defined herein as a polypeptide having alpha-amylase activity that is in its final form following translation and any post-translational modifications, such as N-terminal processing, C-terminal truncation, glycosylation, phosphorylation, etc.
  • improved storage stability refers to an enzyme in accordance with the present disclosure displaying retention of enzymatic activity after a period of incubation relative to wild-type aipha-amylase and/or enzyme not contacted with a stabilizing constituent in accordance with the present disclosure.
  • Suitable enzymes for use in accordance with the present disclosure may be made by cultivating host cells under conditions conducive to the production of one or more enzymes and recovering the enzyme from the cells and/or culture medium.
  • the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the enzyme.
  • Non-limiting suitable media include those available from commercial suppliers and/or media prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).
  • the enzyme secreted from the host cells may be recovered from conventional culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulphate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
  • the stabilizing agent may be added to the enzyme having alpha-amylase activity after the fermentation, recovery and/or purification of the enzyme. Stabilizing constituent
  • the enzyme is stabilized by the presence of a stabilizing constituent in an amount sufficient to stabilize the enzyme.
  • the stabilizing constituent may be a reducing salt selected from the group consisting of sulfites (SO 3 2" ), bisulfites (HSO 3” ), meta bisulfites (pyrosulfites, S 2 O 5 2" ), hydrosulfites (S 2 O 4 2” ), thiosulfates (S 2 O 3 2 ), nitrites (NO 2 " ), phosphites (H 2 PO 3 " , HPO 3 2” ) and hypophosphites (H 2 PO 2 " ).
  • the stabilizing constituent may be a sulfite, a nitrite, a phosphite, or a combinations thereof.
  • examples include: salts of sulfurous acid, salt of pyrosulfurous acid, bisulfite, meta bisulfite, hydrosulfite, hydrogen sulfite or combinations thereof; alkaline earth metal sulfites such as beryllium sulfite, calcium sulfite, magnesium sulfite, strontium sulfite, or combinations thereof; alkali metal sulfites such as lithium sulfite, sodium sulfite, potassium sulfite, or combinations thereof; and/or other sulfites such as sodium pyrosulfite, potassium pyrosulfit, barium sulfite, manganese(ll) sulfite, iron(ll) sulfite, cobalt(ll) sulfite, nickel(ll) sulfite, copper(l
  • the stabilizing constituent in accordance with the present disclosure may be any suitable sulfite that will prevent and/or reduce the degradation enzymes having alpha-amylase activity which may result in a higher proportion of active enzyme being maintained in the enzyme composition during storage. Further, the stabilizing constituent in accordance with the present disclosure may be any suitable sulfite that will provide enzyme compositions with improved storage stability.
  • Suitable nitrites include: salts of nitrous acid; alkaline earth metal nitrites such as beryllium nitrite, calcium nitrite, magnesium nitrite, strontium nitrite, or combinations thereof; alkali metal nitrites such as lithium nitrite, sodium nitrite, potassium nitrite, and/or combinations thereof.
  • the stabilizing constituent in accordance with the present disclosure may be any suitable nitrite that will prevent and/or reduce the degradation enzymes having alpha- amylase activity which may result in a higher proportion of active enzyme being maintained in the enzyme composition during storage. Further, the stabilizing constituent in accordance with the present disclosure may be any suitable nitrite that will provide enzyme compositions with improved storage stability.
  • Suitable phosphites include: salts of phosphorous acid; alkaline earth metal phosphites such as beryllium phosphite, calcium phosphite, magnesium phosphite, strontium phosphite, and/or combinations thereof; alkali metal phosphites such as lithium phosphite, sodium phosphite and potassium phosphite, or combinations thereof.
  • the stabilizing constituent in accordance with the present disclosure may be any suitable phosphite that will prevent or reduce the degradation enzymes having alpha-amylase activity which may result in a higher proportion of active enzyme being maintained in the enzyme composition during storage. Further, the stabilizing constituent in accordance with the present disclosure may be any suitable phosphite that will provide enzyme compositions with improved storage stability.
  • the stabilizing constituent may be added to a liquid composition in liquid or solid form. If the stabilizing constituent is added in liquid form it may be in the form of an aqueous liquid.
  • enzyme compositions for use in accordance with the present disclosure contain one or more stabilizing constituents in an effective amount to improve stability and/or extend shelf life.
  • effective amount refers to an amount of a stabilizing constituent in accordance with the present disclosure sufficient to induce a particular positive benefit to stability or shelf life of enzyme composition in accordance with the present disclosure.
  • the positive benefit can be cosmetic in nature, or activity-related, or a combination of the two.
  • formulations in accordance with the present disclosure may have improved storage stability.
  • retention of enzymatic activity after a period of incubation at elevated temperature may be 2 times, 3, times, 4, times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times longer than a wild-type alpha-amylase and/or enzyme not contacted with a stabilizing constituent in accordance with the present disclosure.
  • stressed conditions include, inter alia, storage such as at an elevated temperature of 4O 0 C for a predetermined period of time, such as 13 weeks.
  • the positive benefit is achieved by contacting enzymes having alpha-amylase activity with a combination of stabilizing constituents, to improve the stability and/or shelf life of a liquid enzyme composition.
  • stable refers to the ability of a material or composition to remain unchanged in enzymatic activity in the presence of heat, moisture or air. With respect to shelf life the terms further can refer to compositions that when in a closed container, remain within predetermined tolerances and limits.
  • examples of acceptable tolerances include enzyme compositions formulated to provide enzyme that retains at least 60%, 70%, 80%, 90%, 95% or 99% of its initial alpha-amylase activity at ambient temperature and/or room temperature for at least about 13 weeks after forming the composition. Those skilled in the art will readily be able to identify the tolerances and limits for other compositions containing enzymes having alpha- amylase activity.
  • Non-limiting examples of other acceptable tolerances include enzyme(s) that retain at least 60%, 70%, 80%, 90%, 95% or 99% of their initial alpha-amylase activity at 4O 0 C for at least about 4 weeks after forming the composition.
  • Another tolerance includes enzymes that retain at least about 30% of their initial alpha-amylase activity at 4O 0 C for at least about 90 days after forming the composition.
  • the particular amount of stabilizing constituent applied generally depends on the purpose for which the composition is to be applied. For example, the amount of stabilizing constituent can vary depending upon the type of enzyme used, concentration of the enzyme, the initial activity of the enzyme, time at which it is applied (pre or post mixture of enzyme), severity of the stability and/or storage problems in a composition and/or type of composition (e.g.
  • one or more stabilizing constituents are applied to an enzyme composition such that the stabilizing constituent is present in an amount of 0.01%-20% by weight of the total composition. In embodiments, one or more stabilizing constituents are present in an amount of about 0.5 to 10% by weight of the total composition. In embodiments, one or more stabilizing constituents are present in an amount of about 0.2 to 0.4% by weight of the total composition. In embodiments, one or more stabilizing constituents are present in an amount of about 0.5 to 5.0% by weight of the total composition. In embodiments, one or more stabilizing constituents are present in an amount of about 0.01% to about 10% by weight of the total composition. In embodiments, one or more stabilizing constituents are present in an amount of about 0.1 % to about 0.6% by weight of the total composition. Enzymes
  • the enzyme to be stabilized may be an alpha-amylase (EC 3.2.1.1 ) or an alternansucrase (EC 2.4.1.140), i.e. a polypeptide having alpha-amylase or altemansucrase activity.
  • the EC numbers are described in the handbook Enzyme Nomenclature from NC- IUBMB, 1992), or at the ENZYME site at the internet: http://www.expasy.ch/enzyme/. It is to be understood that enzyme variants (produced, for example, by recombinant techniques) are included within the meaning of the term "enzyme”. Examples of such enzyme variants are disclosed, e.g. in EP 251,446 (Genencor), WO 91/00345 (Novo Nordisk), EP 525,610 (Solvay) and WO 94/02618 (Gist-Brocades NV).
  • Alpha-amylase The alpha-amylase is included by the enzyme classification EC 3.2.1.1 as set out by the
  • the alpha-amylase may be derived from any organism such as those of animal, fungal, bacterial, or plant origin, e.g. from the genus Absidia, Acremonium, Aspergillus, Coniochaeta, Coriolus, Cryptosporiopsis, Dichotomocladium, Dinemasporium, Diplodia, Fusarium, Gliocladium, Malbranchea, Meripilus, Necteria, Penicillium, Rhizomucor, Stereum, Streptomyces, Subulispora, Syncephalastrum, Thamindium, Thermoascus, Thermomyces, Trametes, Trichophaea, or Valsaria.
  • the alpha-amylase may be obtainable from the species Aspergillus niger, Aspergillus oryzae, or Aspergillus kawachii.
  • suitable alpha-amylases include those obtainable from the species Bacillus licheniformis, Bacillus amybliquefaciens, or Bacillus stearothermophilus.
  • suitable alpha-amylases include maltopentaose-forming alpha-amylases such as those derived from microorganisms belonging to the genus Pseudomonas, see e.g., alpha-amylases described in U.S. Patent No. 6,087,147.
  • alpha-amylase examples include those from pancreatic concentrates of bovine or porcine origin, as well as alpha-amylases manufactured by pharmaceutical companies.
  • plant alpha-amylase and fungal alpha-amylase are acceptable for use in accordance with the present disclosure.
  • plant alpha-amylases include those enzymes from monocotyledonous plants, particularly the cereals barley and rice, as well as dicot alpha-amylases.
  • suitable alpha-amylases for use in accordance with the present disclosure include those described in WO 2006/069290 entitled Enzymes for starch processing, filed 22 December 2005 by Fukuyama et al. (herein incorporated by reference in its entirety), e.g., hybrid alpha-amylase in Table 5 therein, including but not limited to, hybrid alpha-amylase of Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD disclosed as V039 in Table 5 in WO 2006/069290 (Novozymes A/S).
  • suitable alpha-amylase for use in accordance with the present disclosure include alpha-amylase from species of the Genus Pyrococcus, such as e.g., Pyrococcus furiosus as disclosed in WO 94/19454.
  • alpha-amylase suitable for use in accordance with the present disclosure include Bacillus alpha-amylases, such as a Termamyl-like alpha-amylase, which including the B. licheniformis alpha-amylase (Commercially available as TERMAMYL®
  • stearothermophilus alpha-amylase sold as TERMAMYL® (Novozymes A/S 102 L type S), the alpha-amylases derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB
  • Alpha-amylases with the definition of "Termamyl-like alpha-amylase” are described in for instance WO 96/23874 (Novozymes A/S). Further, Termamyl-like alpha-amylase is an alpha-amylase as described in WO99/19467 on page 3, line 18 to page 6, line 27.
  • the alpha-amylase is a recombinant ⁇ . stearothermophilus alpha-amylase variant with the mutations: I181*+G182 * +N193F.
  • non-limiting examples of suitable enzymes having alpha-amylase activity include Fungamyl-like alpha-amylases including FUNGAMYL® (from Novozymes, Denmark).
  • FUNGAMYL® is a fungal alpha-amylase obtained from a selected strain of Aspergillus oryzae.
  • Such fungamyl-like amylases that may be contacted with stabilizing constituent in accordance with the present disclosure are further described in U.S. Patent Publication No. 20070128312 assigned to Novozymes A/S.
  • alpha-amylases include CLARASETM (from Genencor Int., USA) derived from Aspergillus oryzae; and MALTAMYLTM (from Enzyme Biosystems) derived from Aspergillus niger.
  • suitable enzymes having alpha-amylase activity include acid alpha- amylase, such as those of fungal or bacterial origin.
  • the term "acid alpha-amylase” means an alpha-amylase (E.C. 3.2.1.1) which added in an effective amount has activity optimum at a pH in the range of 2 to 7, preferably from 3 to 6, or more preferably from 3.5-5.5.
  • suitable enzymes having alpha-amylase activity include bacterial alpha-amylases.
  • the bacterial alpha-amylases may be derived from the genus Bacillus.
  • the Bacillus alpha-amylase is derived from a strain of S. licheniformis, B. amyloliquefaciens, B. subtilis or S. stearothermophilus, but may also be derived from other Bacillus sp.
  • contemplated alpha-amylases include the Bacillus licheniformis alpha-amylase (BLA) shown in SEQ ID NO: 4 in WO 99/19467, the Bacillus amyloliquefaciens alpha-amylase (BAN) shown in SEQ ID NO: 5 in WO 99/19467, and the Bacillus stearothermophilus alpha-amylase (BSG) shown in SEQ ID NO: 3 in WO 99/19467.
  • BLA Bacillus licheniformis alpha-amylase
  • BAN Bacillus amyloliquefaciens alpha-amylase
  • BSG Bacillus stearothermophilus alpha-amylase
  • the enzymes having alpha-amylase activity include enzyme having a degree of identity of at least 60%, or at least 70%, or at least 80%, or at least 90%, such as at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to any of the sequences shown as SEQ ID NOS: 1 , 2, 3, 4, or 5, respectively, in WO 99/19467.
  • the Bacillus alpha-amylase may also be a variant and/or hybrid, especially one described in any of WO 96/23873, WO 96/23874, WO 97/41213, WO 99/19467, WO 00/60059, and WO 02/10355 (all documents hereby incorporated by reference in their entirety).
  • WO 96/23873 WO 96/23874
  • WO 97/41213 WO 99/19467
  • WO 00/60059 WO 02/10355
  • Specifically contemplated alpha-amylase variants are disclosed in US patent nos. 6,093,562, 6,297,038 or US patent no.
  • BSG alpha-amylase Bacillus stearothermophilus alpha-amylase (BSG alpha-amylase) variants having a deletion of one or two amino acid in position 179 to 182, preferably a double deletion disclosed in WO 1996/023873 - see e.g., page 20, lines 1-10 (hereby incorporated by reference), preferably corresponding to delta(181-182) compared to the wild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO: 3 disclosed in WO 99/19467 or deletion of amino acids 179 and 180 using SEQ ID NO: 3 in WO 99/19467 for numbering (which reference is hereby incorporated by reference).
  • BSG alpha-amylase Bacillus stearothermophilus alpha-amylase
  • alpha-amylase include Bacillus alpha-amylases, especially Bacillus stearothermophilus alpha-amylase, which have a double deletion corresponding to delta(181-182) and further comprise a N193F substitution (also denoted 1181* + G182* + N193F) compared to the wild-type BSG alpha-amylase amino acid sequence set forth in SEQ ID NO: 3 disclosed in WO 99/19467.
  • the enzymes having alpha-amylase activity include maltogenic alpha-amylase.
  • a "maltogenic alpha-amylase” (glucan 1 ,4-alpha-maltohydrolase, E. C. 3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose in the alpha-configuration.
  • a maltogenic alpha- amylase from Bacillus stearothermophilus strain NCIB 11837 is commercially available from Novozymes A/S, Denmark.
  • the maltogenic alpha-amylase is described in US patent nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby incorporated by reference in their entirety.
  • suitable alpha-amylases and/or enzymes having alpha-amylase activity include bacterial hybrid alpha-amylases.
  • the hybrid alpha-amylase specifically contemplated comprises 445 C-terminal amino acid residues of the Bacillus licheniformis alpha-amylase (shown as SEQ ID NO: 4 in WO 99/19467) and the 37 N-terminal amino acid residues of the alpha-amylase derived from Bacillus amyloliquefaciens (shown as SEQ ID NO: 3 in WO 99/194676), with one or more, especially all, of the following substitution: G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using the Bacillus licheniformis numbering).
  • variants having one or more of the following mutations (or corresponding mutations in other Bacillus alpha-amylase backbones): H154Y, A181T, N 190F, A209V and Q264S and/or deletion of two residues between positions 176 and 179, preferably deletion of E178 and G179 (using the SEQ ID NO: 5 numbering of WO 99/19467).
  • the bacterial alpha-amylase may be added in amounts well-known in the art.
  • suitable alpha-amylases or enzymes having alpha-amylase activity include one or more acid fungal alpha-amylases.
  • Acid fungal alpha-amylases include acid alpha- amylases derived from a strain of the genus Aspergillus, such as Aspergillus oryzae, Aspergillus niger, and Aspergillus kawachii.
  • acid fungal alpha-amylase is a Fungamyl-like alpha-amylase which is derived from a strain of Aspergillus oryzae.
  • Fungamyl-like alpha- amylase includes an alpha-amylase which exhibits a high identity, i.e.
  • suitable acid alpha-amylase and/or enzymes having alpha-amylase activity include those derived from a strain Aspergillus niger.
  • the acid fungal alpha-amylase is the one from A. niger disclosed as "AMYA_ASPNG" in the Swiss-prot/TeEMBL database under the primary accession no. P56271 and described in more detail in WO 89/01969 (Example 3).
  • the acid Aspergillus niger acid aipha-amylase is also shown as SEQ ID NO: 1 in WO 2004/080923 (Novozymes) which is hereby incorporated by reference. Also variants of said acid fungal amylase having at least 70% identity, such as at least 80% or even at least 90% identity, such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 1 in WO 2004/080923 are contemplated.
  • a suitable commercially available acid fungal alpha-amylase derived from Aspergillus niger is SP288 (available from Novozymes A/S, Denmark).
  • the alpha-amylase or enzyme having alpha-amylase activity is derived from Aspergillus kawachii and disclosed by Kaneko et al. J. Ferment. Bioeng. 81:292-298(1996) "Molecular-cloning and determination of the nucleotide-sequence of a gene encoding an acid- stable alpha-amylase from Aspergillus kawachii.”; and further as EMBL:#AB008370.
  • the fungal acid alpha-amylase may also be a wild-type enzyme comprising a carbohydrate-binding module (CBM) and an alpha-amylase catalytic domain (i.e., a none- hybrid), or a variant thereof.
  • CBM carbohydrate-binding module
  • alpha-amylase catalytic domain i.e., a none- hybrid
  • the wild-type acid alpha-amylase is derived from a strain of Aspergillus kawachii.
  • suitable alpha-amylases or enzymes having alpha-amylase activity for use in accordance with the present disclosure include one or more fungal hybrid alpha- amylases.
  • the fungal acid alpha-amylase is a hybrid alpha-amylase.
  • Non- limiting examples of fungal hybrid alpha-amylases include those disclosed in WO 2005/003311 or U.S. Patent Publication no. 2005/0054071 (Novozymes) or US patent application no. 60/638,614 (Novozymes) which is hereby incorporated by reference in its entirety.
  • a hybrid alpha-amylase may comprise an alpha-amylase catalytic domain (CD) and a carbohydrate- binding domain/module (CBM) and optionally a linker.
  • Non-limiting examples of contemplated hybrid alpha-amylases include those disclosed in U.S. patent application no.
  • 60/638,614 including Fungamyl variant with catalytic domain JA118 and Athelia rolfsii SBD (SEQ ID NO: 100 in U.S. application no. 60/638,614), Rhizomucor pusillus alpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ ID NO: 101 in U.S. application no. 60/638,614) and Meripilus giganteus alpha-amylase with Athelia rolfsii glucoamylase linker and SBD (SEQ ID NO: 102 in U.S. application no. 60/638,614).
  • hybrid alpha-amylase consisting of Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD disclosed as V039 in Table 5 of Example 4 in WO 2006/069290 (Novozymes A/S) are suitable for use in accordance with the present disclosure and may be contacted with stabilizing constituent in accordance with the present disclosure.
  • This hybrid alpha-amylase may be further combined with glucoamylase, such as that derived from Trametes cingulata disclosed in SEQ ID NO: 2 in WO 2006/069289 to form enzyme mixtures.
  • glucoamylase from Asp. Niger may be combined with this mixture of enzymes.
  • contemplated hybrid alpha-amylases for use in accordance with the present disclosure include those disclosed in U.S. Patent Publication no.
  • suitable alpha-amylases for use in accordance with the present disclosure include one or more commercial alpha-amylase products.
  • Non-limiting examples of commercial products comprising alpha-amylase include MYCOLASETM from DSM (Gist Brocades), BANTM, DURAMYLTM, TERMAMYLTM SC, FUNGAMYLTM, LIQUOZYMETM X and SANTM SUPER, SANTM EXTRA L, NATALASETM, STAINZYMETM, STAINZYMETM Ultra (Novozymes A/S) and CLARASETM L-40,000, DEX-LOTM, SPEZYMETM FRED, SPEZYMETM AA, SPEZYMETM HPA and SPEZYMETM DELTA AA (Genencor Int.), and the acid fungal alpha- amylase sold under the trade name SP288 (available from Novozymes A/S, Denmark).
  • a suitable alpha-amylase for use in accordance with the present disclosure is the mature part of the alpha-amylase disclosed in Richardson et al. (2002), The Journal of Biological Chemistry, Vol. 277, No 29, Issue 19 July, pp. 267501-26507, referred to as
  • BD5088 This alpha-amylase is the same as the one shown in SEQ ID NO: 2. The mature enzyme sequence starts after the initial "Met” amino acid in position 1.
  • alpha-amylase suitable for use in accordance with the present disclosure includes those expressed in Aspergillus kawachii such as those described in WO2005118795, WO2005118800, WO2005117953 and WO2005117756.
  • alpha-amylase used in accordance with the present disclosure is derived from a microorganism, preferably a bacterium, of the order Thermococcales.
  • the alpha-amylase may be a hybrid alpha-amylase such as the BD5088 alpha- amylase made from alpha-amylases from three microorganisms within the order Thermococcales.
  • Suitable alpha-amylases for use in accordance with the present disclosure also include amino acid sequences having at least 60%, at least 65%, at least 70%, at least 75%, at least
  • the degree of identity between two amino acid sequences is determined by the Clustal method (Higgins, 1989, CABIOS 5: 151-153) using the LASERGENETM MEGALIGNTM software (DNASTAR, Inc., Madison, Wl) with an identity table and the following multiple alignment parameters: Gap penalty of 10 and gap length penalty of 10.
  • the alpha-amylase amino acid sequence has an amino acid sequence which differs from any of the aforementioned amino acid sequences in no more than 10 positions, no more than 9 positions, no more than 8 positions, no more than 7 positions, no more than 6 positions, no more than 5 positions, no more than 4 positions, no more than 3 positions, no more than 2 positions, or even no more than 1 position.
  • Alpha-amylase enzymes employed in compositions in accordance with the present disclosure will depend on a number of factors including, but not limited to the nature of the alpha-amylase, the concentration of the alpha-amylase, the nature of any solvents present and the nature of the ultimate product to be formulated using the composition.
  • alpha-amylase is present in an amount effective to provide alpha-amylase activity.
  • the one or more enzyme(s) having alpha-amylase activity is present in an amount of about 0.1% to about 30% by weight of the total composition, in some embodiments in an amount of about 1.0% to about 20% by weight of the total composition, and in some embodiments in an amount of about 1.0% to about 10% by weight of the total composition.
  • an acid alpha-amylases according to the present disclosure may be added to a composition and/or formulation in an amount of 0.1 to 10 AFAU/g DS, or 0.10 to 5 AFAU/g DS, or 0.3 to 2 AFAU/g DS or 0.001 to 1 FAU-F/g DS, or 0.01 to 1 FAU-F/g DS.
  • the alpha-amylase activity when measured in KNU units is present in compositions in an amount of 0.0005-5 KNU per g DS, or 0.001-1 KNU per g DS, such as around 0.050 KNU per g DS.
  • the alpha-amylase when measured in FAU/F units is preferably present in compositions in an amount of 5 to 1000 FAU/F/g, and in embodiments, 10 to about 150 FAU/F/g, and in embodiments, 40 to 44 FAU/F/g, and in embodiments, about 10 to 40 FAU/F/g.
  • Alpha-Amylase Sequence 5 to 1000 FAU/F/g, and in embodiments, 10 to about 150 FAU/F/g, and in embodiments, 40 to 44 FAU/F/g, and in embodiments, about 10 to 40 FAU/F/g.
  • Catalytic domains i.e., alpha-amylase catalytic domains (in particular acid stable alpha-amylases), which are appropriate for construction of polypeptides of the types of the present disclosure may be derived from any organism, preferred are those of fungal or bacterial origin.
  • the alpha-amylase is a wild type enzyme.
  • the alpha- amylase is a variant alpha-amylases comprising amino acid modifications leading to increased activity, increased protein stability at low pH, and/or at high pH, increased stability towards calcium depletion, and/or increased stability at elevated temperature.
  • alpha-amylases for use in a hybrid embodiment include alpha- amylases obtainable from a species selected from the list consisting of Absidia, Acremonium, Aspergillus, Coniochaeta, Coniochaeta, Cryptosporiopsis, Dichotomocladium, Dinemasporium sp., Diplodia, Fusarium, Gliocladium, Malbranchea, Meripilus Trametes, Nectria, Nectria, Penicillium, Phanerochaete, Rhizomucor, Rhizopus, Streptomyces, Subulispora, Syncephalastrum, Thaminidium, Thermoascus, Thermomyces, Trametes, Trichophaea and Valsaria.
  • the alpha-amylases catalytic domain may also be derived from a bacteria, e.g., Bacillus.
  • the alpha-amylases amino acid sequence selected is derived from any species selected from the group consisting of Absidia cristata, Acremonium sp., Aspergillus niger, Aspergillus kawachii, Aspergillus oryzae, Coniochaeta sp., Coniochaeta sp., Cryptosporiopsis sp., Dichotomocladium hesseltinei, Dinemasporium sp., Diplodia sp., Fusarium sp., Gliocladium sp., Malbranchea sp., Meripilus giganteus, Nectria sp., Nectria sp., Penicillium sp., Phanerochaete chrysosporium, Rhizomucor pusillus, Rhizopus oryzae, Stereum sp.
  • Streptomyces thermocyaneoviolaceus Streptomyces limosus, Subulispora procurvata, Syncephalastrum racemosum, Thaminidium elegans, Thermoascus aurantiacus, Thermoascus sp., Thermomyces lanuginosus, Trametes corrugata, Trametes sp., Trichophaea saccata, Valsaria rubricosa, Valsaria spartii and Bacillus flavothermus (Syn. Anoxybacillus contaminans).
  • composition and methods of the present disclosure are particularly useful for alpha-amylases
  • one or more secondary enzymes may be combined with the alpha-amylases in accordance with the present disclosure to form stable enzyme mixtures and/or stable product formulations.
  • Non-limiting examples of secondary enzymes in accordance with the present disclosure include oxidoreductases (EC 1.-.-.-), transferases (EC 2.-.-.-), hydrolases (EC 3.-.-.-), lyases (EC 4.-.-.-), isomerases (EC 5.-.-.-), ligases (EC 6.-.-.-) and combinations thereof.
  • Conventional alpha-amylases (EC 3.2.1.1)(such as those alpha-amylases not contacted with a stabilizing constituent in accordance with the present disclosure) can also be included herewith.
  • carbohydrases e.g. a second ⁇ -amylase (EC 3.2.1.1).
  • carbohydrases e.g. a second ⁇ -amylase (EC 3.2.1.1).
  • carbohydrase is used to denote not only enzymes capable of breaking down carbohydrate chains (e.g. starches or cellulose) of especially five- and six-membered ring structures (i.e. glycosidases, EC 3.2), but also enzymes capable of isomerizing carbohydrates, e.g. six-membered ring structures such as D-glucose to five-membered ring structures such as D-fructose.
  • Non-limiting carbohydrases of relevance include the following (EC numbers in parentheses): ⁇ -amylases (EC 3.2.1.2), glucan 1 ,4- ⁇ -glucosidases (EC 3.2.1.3), endo-1 ,4-beta- glucanase (cellulases, EC 3.2.1.4), endo-1 ,3(4 )- ⁇ -glucanases (EC 3.2.1.6), endo-1 ,4- ⁇ - xylanases (EC 3.2.1.8), dextranases (EC 3.2.1.11), chitinases (EC 3.2.1.14), polygalacturonases (EC 3.2.1.15), iysozymes (EC 3.2.1.17), ⁇ -glucosidases (EC 3.2.1.21), ⁇ -galactosidases (EC 3.2.1.22), ⁇ -galactosidases (EC 3.2.1.23), amylo-1 ,6-glucos
  • the secondary enzymes may be a pullulanase (EC 3.2.1.41) such as pullulan-6- glucanohydrolase.
  • a glucoamylase (EC 3.2.1.3) may be combined with the alpha-amylases of the present disclosure to form a composition or formulation.
  • glucoamylase derived from Trametes cingulata disclosed in SEQ ID NO: 2 in WO 2006/069289 (herein incorporated by reference in its entirety) and available from Novozymes A/S may be combined with the alpha- amylases of the present disclosure to form a composition.
  • one or more glucoamylases can be combined with the alpha-amylase in accordance with the present disclosure, e.g. conventional glucoamylase derived from Asp. Niger in combination with glucoamylase derived from Trametes cingulata disclosed in SEQ ID NO: 2 in WO 2006/069289.
  • hybrid alpha-amylase consisting of Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD disclosed as V039 in Table 5 of Example 4 in WO 2006/069290 (Novozymes A/S) contacted with stabilizing constituent in accordance with the present disclosure, may be combried with glucoamylase, such as that derived from Trametes cingulata disclosed in SEQ ID NO: 2 in WO 2006/069289.
  • glucoamylase from Asp. Niger may be combined with this mixture of enzymes.
  • the secondary enzyme constituent is present in an amount of about 0.1% to 30% by weight of the total composition, and in some embodiments in an amount of about 1.0% to about 20% by weight of the total composition, and in some embodiments in an amount of about 1.0% to about 10% of the total composition.
  • an alternansucrase enzyme may be contacted with stabilizing constituent in accordance with the present disclosure.
  • the stabilizing constituent may be present in an amount sufficient to reduce the degradation of the alternansucrase activity and/or maintain its initial activity.
  • the stabilizing constituent may be present in an amount of about 0.01% to about 20% by weight of the total composition.
  • the enzyme compositions may be formulated to comprise enzyme that retains at least 60%, 70%, 80% or 90% of its initial enzyme activity at a temperature of 4O 0 C temperature for at least about 4 weeks after forming the composition.
  • the alternansucrase is present in an amount of about 0.1% to 30% by weight of the total composition, and in some embodiments in an amount of about 1.0% to about 20% by weight of the total composition, and in some embodiments in an amount of about 1.0% to about 10% of the total composition.
  • the salt stabilizing constituent is selected from the group consisting of NaNO 3 , K 2 SO 3 and combinations thereof.
  • the stabilizing constituent is an aqueous solution of one or more water soluble salts including sulfite ion (SO 3 2" ), bisulfite ion (HSO 3" ), meta bisulfite ion (S 2 O 5 2" ) and hydrosulfite ion (S 2 O 4 2" ) and combinations thereof.
  • Products containing enzyme having alpha-amylase activity in accordance with the present disclosure can be in the form of solid compositions, solutions, emulsions (including microemulsions), gels, powders, or other typical solid or liquid compositions used for enzyme application.
  • Such products may include enzyme having alpha-amylase activity in accordance with the present disclosure and one or more secondary enzymes.
  • the products may include the enzymes of the present disclosure in combination with other granule forming constituents.
  • granule forming constituents include inert cores, additional granulation materials, such as binders, polysaccharides, synthetic polymers, waxes, enhancing agents, fillers, fiber materials, enzyme stabilizing agents, solubilizing agents, crosslinking agents, suspension agents, viscosity regulating agents, light spheres, chlorine scavengers, plasticizers, pigments, salts, lubricants (such as surfactants or antistatic agents) and fragrances.
  • WO 2006/053564 entitled Stabilization of Granules Comprising Active Compounds filed on 17 November 2005 (herein incorporated by reference in its entirety). It is envisioned that the solid granules described in WO 2006/053564 can be modified to include the stable enzymes having alpha-amylase activity in accordance with the present disclosure.
  • Liquid products containing enzyme having alpha-amylase activity in accordance with the present disclosure can be in the form of solutions or liquids.
  • Such products may include enzyme having alpha-amylase activity in accordance with the present disclosure, one or more secondary enzymes, and additional materials such as buffers, preservatives, stabilizing agents, solubilizing agents, crosslinking agents, suspension agents, viscosity regulating agents, chlorine scavengers, pigments, salts, lubricants (such as surfactants or antistatic agents) and fragrances.
  • the pH of the liquid composition can be adjusted using a pH adjustor to an amount of 4 to 7.
  • stabilized enzyme compositions in accordance with the present disclosure have a pH above 5, above 5.5, above 6, above 6.5, above 6.6, above 6.7 and still maintain excellent stability as described herein.
  • stabilized liquid enzyme compositions in accordance with the present disclosure have a pH in the range of 3.5 to 11 , and in some embodiments a pH of 4.0 to 7, and in some embodiments a pH of about 4.5 to 5.
  • the stabilized enzymes in accordance with the present disclosure possess valuable properties allowing for a variety of industrial applications.
  • Stabilized enzyme of the present disclosure are applicable as a component in raw starch hydrolysis, washing, dishwashing and hard-surface cleaning compositions.
  • Numerous stabilized enzymes are particularly useful in the production of sweeteners and ethanol from starch, and/or for textile desizing.
  • Conditions for conventional starch-conversion processes, including starch liquefaction and/or saccharification processes, are described in, e.g., U.S. Pat. No. 3,912,590 and in EP patent publications Nos. 252,730 and 63,909.
  • enzymes having alpha-amylase activity of the present disclosure may suitably be incorporated in detergent compositions.
  • Detergent compositions comprising enzyme having alpha-amylase activity of the present disclosure may additionally comprise one or more other enzymes, such as a lipase, cutinase, protease, cellulase, peroxidase or laccase, and/or another alpha-amylase.
  • Alpha-amylase enzymes of the present disclosure may be incorporated in detergents at conventionally employed concentrations. It is at present contemplated that an alpha-amylase enzyme of the present disclosure may be incorporated in an amount corresponding to 0.00001-1 mg (calculated as pure, active enzyme protein) of alpha-amylase per liter of wash/dishwash liquor using conventional dosing levels of detergent.
  • alpha-amylase activity may be expressed in "Fungal alpha-Armylase Units" (FAU).
  • FAU is the amount of enzyme which under standard conditions (i.e. at 37 0 C and pH 4.7) breaks down 5260 mg solid starch (Amylum solubile, Merck) per hour.
  • a folder AF 9.1/3, describing this FAU assay in more details, is available upon request from Novozymes A/S, Denmark, which folder is hereby included by reference.
  • alpha-amylase activity may be expressed in
  • FAU(F) or Fungal Alpha-Amylase Units Fungamyl.
  • BAN Bacterial amylase
  • ⁇ -glucosidase in samples and the enzyme of ⁇ -glucosidase in the reagent kit hydrolyze substrate (4,6-ethylidene (G 7 )-p-nitrophenyl (Gi)- ⁇ , D-maltoheptaoside (ethylidene-G 7 PNP) to glucose and the yellow- colored p-nitrophenol.
  • the rate of formation of p-nitrophenol can be observed by Konelab 30. This is an expression of the reaction rate and thereby the enzyme activity.
  • FAU(F) Fungal Alpha-Amylase LJnits (Fungamyl) is measured relative to an enzyme standard of a declared strength.
  • AFAU Acid Fungal Alpha-amylase Units
  • 1 AFAU is defined as the amount of enzyme which degrades 5.260 mg starch dry matter per hour under the below mentioned standard conditions.
  • Acid alpha-amylase i.e., acid stable alpha-amylase, an endo-alpha-amylase (1,4-alpha-
  • D-glucan-glucano-hydrolase hydrolyzes alpha-1 ,4-glucosidic bonds in the inner regions of the starch molecule to form dextrins and oligosaccharides with different chain lengths.
  • the intensity of color formed with iodine is directly proportional to the concentration of starch.
  • Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under the specified analytical conditions.
  • Iodine (I2) 0.03 g/L
  • KNU Alpha-amylase activity
  • the alpha-amylase activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which is compared to a colored glass standard.
  • KNU Kilo Novo alpha-amylase Unit
  • Composition A included a mixture of two enzymes.
  • Enzyme 1 was a hybrid alpha-amylase consisting of Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD disclosed as V039 in Table 5 in WO 2006/069290 (Novozymes A/S).
  • Enzyme 1 was contacted with sodium sulfite as a stabilizing constituent.
  • Enzyme 2 was a Glucoamylase derived from Trametes cingulata disclosed in SEQ ID NO: 2 in WO 2006/069289 and available from Novozymes A/S.
  • Composition A had additional constituents and characteristics as shown in Table 1 below.
  • Composition B included a mixture of three enzymes. Enzyme 1 and Enzyme 2 were the same as in Composition A. Enzyme 3 was a glucoamylase derived from Aspergillus Niger. Composition B had additional constituents and characteristics as shown in Table 1 below. TABLE 1
  • a stabilized formulation was made as follows:
  • Example 4 A stabilized formulation was made as follows:
  • a stabilized formulation was made as follows: 611.1Og of a hybrid alpha-amylase consisting of Rhizomucor pusillus alpha-amylase with Aspergillus niger glucoamylase linker and SBD disclosed as V039 in Table 5 in WO 2006/069290 (Novozymes A/S) having an initial activity of 175.6FAU-F was mixed with 5.96g of Sodium Sulphite. This mixture was further combined with 485.43g of a glucoamylase derived from Trametes cingulata disclosed in SEQ ID NO: 2 in WO 2006/069289 and available from Novozymes A/S. About 1.25g of preservative was added to the mixture along with 396.73g of diluent (mixture of glucose, water).
  • a stabilized formulation was made as follows: 611.1Og of a hybrid alpha-amylase having an initial activity of 175.6FAU-F was mixed with 5.96g of a salt of nitrous acid. About 1.25g of preservative was added to the mixture along with 396.73g of diluent (mixture of glucose, water). Stabilization was expected.
  • a stabilized formulation was made as follows:
  • 611.1Og of a hybrid alpha-amylase having an initial activity of 175.6FAU-F was mixed with 5.96g of a salt of phosporous acid. About 1.25g of preservative was added to the mixture along with 396.73g of diluent (mixture of glucose, water). Stabilization was expected.
  • An alternansucrase enzyme solution was prepared by contacting altemansucrase enzyme with sulfite stabilizing constituent in accordance with the present disclosure.
  • the compositions were placed on stability and the following results were observed: TABLE 3
  • Enzyme compositions were formulated and tested as follows. The same alpha-amylase and glucoamylase as Enzymes 1 and 2 in Example 1 were used.
  • Stabilizing constituent as indicated below

Abstract

L'invention concerne l'amélioration de la stabilité de stockage d'une alpha-amylase comprenant un domaine de liaison aux carbohydrates, par incorporation de compositions qui sont stabilisées contre la décomposition par addition de constituants de stabilisation tels que des sels de sulfite, permettant d'obtenir une durée de conservation supérieure pour les produits fabriqués à l'aide des compositions et/ou une stabilité de stockage améliorée. L'invention concerne également un procédé de stabilisation d'une composition enzymatique.
PCT/EP2008/061695 2007-09-05 2008-09-04 Compositions enzymatiques contenant un constituant de stabilisation WO2009030728A2 (fr)

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WO2011086293A1 (fr) 2009-12-22 2011-07-21 Institut National De La Recherche Agronomique - Inra Sulfatase modifiant selectivement les glycosaminoglycanes
WO2013006756A3 (fr) * 2011-07-06 2013-05-02 Novozymes A/S Variants d'alpha-amylase et polynucléotides codant ces variants
US9267124B2 (en) 2011-07-06 2016-02-23 Novozymes A/S Alpha amylase variants and polynucleotides encoding same
US9765316B2 (en) 2011-07-06 2017-09-19 Novozymes A/S Alpha amylase variants and polynucleotides encoding same
EP2729572B1 (fr) * 2011-07-06 2019-03-20 Novozymes A/S Variants d'alpha-amylase et polynucléotides codant ces variants
CN105369637A (zh) * 2015-12-18 2016-03-02 江南大学 一种纯淀粉上浆棉织物的一浴退浆精练方法
WO2017106633A1 (fr) * 2015-12-18 2017-06-22 Basf Enzymes Llc Formulation liquide d'alpha-amylase
WO2023170628A1 (fr) * 2022-03-09 2023-09-14 Lallemand Hungary Liquidity Management Llc Alpha-amylases bacteriennes et archaéales

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