WO1995023853A1 - Desulfurococcus amylase and pullulanase - Google Patents

Desulfurococcus amylase and pullulanase Download PDF

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Publication number
WO1995023853A1
WO1995023853A1 PCT/DK1995/000098 DK9500098W WO9523853A1 WO 1995023853 A1 WO1995023853 A1 WO 1995023853A1 DK 9500098 W DK9500098 W DK 9500098W WO 9523853 A1 WO9523853 A1 WO 9523853A1
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Prior art keywords
pullulanase
amylase
starch
determined
desulfurococcus
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PCT/DK1995/000098
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French (fr)
Inventor
Carsten SJØHOLM
Garabed Antranikian
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Novo Nordisk A/S
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Priority to EP95910469A priority Critical patent/EP0753056A1/en
Priority to AU17562/95A priority patent/AU1756295A/en
Publication of WO1995023853A1 publication Critical patent/WO1995023853A1/en

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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)
    • C12N9/24—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
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C12N9/2457—Pullulanase (3.2.1.41)
    • 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
    • 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
    • C12N9/2451—Glucanases acting on alpha-1,6-glucosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/16—Preparation of compounds containing saccharide radicals produced by the action of an alpha-1, 6-glucosidase, e.g. amylose, debranched amylopectin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01041—Pullulanase (3.2.1.41)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to a novel thermostable amylase and a novel thermostable pullulanase and their use in 5the production of sweeteners and ethanol from starch.
  • sweeteners from starch has been largely improved by application of different microbial enzymes to obtain better quality and yields, but the necessity of 10performing several steps of the starch-hydrolysing process at elevated temperatures means that there is still a need for new starch-hydrolysing enzymes with increased thermal stability.
  • Pyrococcus e.g. Pyrococcus wosei and Pyrococcus furiosus. for reference see Arch. Microbiol. 15155. 1991, pp. 572-578, and Appl. Env. Microbiol. 56. 1990, pp.1985-1991, can produce highly thermostable amylases.
  • thermostable amylase and a novel thermostable pullulanase can be obtained from Desulfurococcus mucosus. a strain not previously reported to produce thermostable amylase and pullulanase; these new
  • 25enzymes have temperature optimum around 100°C.
  • the invention provides an amylase preparation, characterized by being producible by cultivation of an amylase producing strain of Desulfurococcus mucosus. and a pullulanase preparation, characterized by being producible by
  • Fig. 1 shows the relative activity (% rel. ) of an amylase (°) and a pullulanase ( ⁇ ) of the invention at various temperatures (determined at pH 5.5 with starch and pullulan, respectively, as substrate) .
  • Fig. 2 shows the relative activity (% rel.) of an amylase ( ⁇ ) and a pullulanase ( ⁇ ) of the invention at various pH, determined at 90°C with starch and pullulan, respectively, as substrate.
  • amylase is derived from an amylase producing strain of Desulfurococcus mucosus and pullulanase is derived from a pullulanase producing strain of Desulfurococcus mucosus.
  • a strain representative of Desulfurococcus mucosus has been made publicly available under Accession No. DSM 2162. The number is published in the DSM Catalogue of Strains, 1993.
  • Amylase and pullulanase of the invention may be produced by anaerobic cultivation of the above mentioned strain on a nutrient medium containing suitable carbon and nitrogen sources, such media being known in the art. Anaerobic con ⁇ ditions may be achieved during the preparation of media by sparging with N 2 and following the anaerobic techniques as described by Balch and Wolfe in Appl. Env. Microbiol. 32, 1976, pp. 781-791.
  • amylase and pullulanase of the invention can be produced by aerobic cultivation of a trans ⁇ formed host organism containing the appropriate genetic information from the above mentioned strain. Such transformants can be prepared and cultivated by methods known in the art.
  • the amylase and the pullulanase may be recovered by removing the cells from the fermentation medium (e.g. by centrifugation or filtration) and then concentrating the broth (e.g. by ultrafiltration) . If desired, the amylase and the pullulanase may be further purified by known methods.
  • the enzymes of the invention have immunochemical properties identical or partially identical (i.e. at least partially identical) to those of an enzyme derived from the strain Desulfurococcus mucosus. DSM 2162.
  • the immunochemical properties can be determined immunologically by cross-reaction identity tests.
  • the identity tests can be performed by the well-known Ouchterlony double immunodiffusion procedure or by tandem crossed immunoelectro- phoresis according to Axelsen N.H. ; Handbook of Immunopre- cipitation-in-Gel Techniques; Blackwell Scientific Publications
  • Monospecific antisera are generated according to the above mentioned method by immunizing rabbits with the purified enzymes of the invention.
  • the immunogens are mixed with Freund's adjuvant and injected subcutaneously into rabbits every second week.
  • Antisera are obtained after a total im ⁇ munization period of 8 weeks, and immunoglobulins are prepared therefrom as described by Axelsen N.H. , supra.
  • An amylase of the invention can be characterized by having amylase activity at temperatures of from below 60°C to approximately 120°C, having activity optimum at temperatures in the range 95-105°C, determined at pH 5.5 with starch as substrate.
  • the amylase can also be characterized by having amylase activity at pH values of from below pH 4.0 to ap- proximately pH 11.0, having optimum in the range pH 5.5 to pH 6.5, determined at 90°C with starch as substrate.
  • a pullulanase of the invention can be characterized by having pullulanase activity at temperatures of from below 60°C to approximately 120°C, having activity optimum at temperatures in the range 90-105°C, determined at pH 5.5 with pullulan as substrate.
  • the pullulanase can also be charac ⁇ terized by having pullulanase activity at pH values of from below pH 4.0 to approximately pH 9.0, having optimum in the range pH 4.8 to pH 5.8, determined at 90°C with pullulan as substrate.
  • Amylase activity is determined by measuring the amount of reducing sugar released during the incubation with starch.
  • One unit (U) of amylase activity is defined as the amount of amylase that releases 1 ⁇ mole of reducing sugar (as maltose standard) per min. under the following assay con ⁇ ditions: A 0.05 ml volume of 1% soluble starch is added to 0.05 ml of 0.1 M sodium acetate buffer pH 5.5. 25 ⁇ l of enzyme solution are added to this mixture and the sample is incubated at 90°C for 30 min. The reaction is stopped by cooling on ice, and the amount of reducing sugar is determined by dinitro- salicylic acid. Sample blanks are used to correct for non- enzymatic release of reducing sugar.
  • Pullulanase activity is determined by measuring the amount of reducing sugar released during the incubation with pullulan.
  • One unit (U) of pullulanase activity is defined as the amount of pullulanase that releases 1 ⁇ mole of reducing sugar (as maltose standard) per min. under the following assay conditions: A 0.05 ml volume of 1% pullulan is added to 0.05 ml of 0.1 M sodium acetate buffer pH 5.5. 25 ⁇ l of enzyme solution are added to this mixture and the sample is incubated at 90°C for 30 min. The reaction is stopped by cooling on ice, and the amount of reducing ⁇ sugar is determined by dinitrosalicylic acid. Sample blanks are used to correct for nonenzymatic release of reducing sugar.
  • the enzymes of this invention possess valuable properties allowing for various industrial applications.
  • the enzymes in being thermostable, find potential application in the production of sweeteners and ethanol from starch.
  • Conditions for conventional starch converting processes and liquefaction and/or saccharification processes are de ⁇ scribed in for instance US Patent No. 3,912,590 and EP patent publications Nos. 252,730 and 63,909.
  • DSM 2162 The strain Desulfurococcus mucosus, DSM 2162, was recultured from glycerol-preserved cells using the medium recommended by the Deutsche Sammlung von Mikroorganismen (DSM) .
  • the microorganisms were grown in 1 liter batch cultures under the following conditions: Medium: DSM184 (DSM184 is described in DSM Catalogue of Strains, 1993) , pH 5.8, temp. 85°C; in the medium sulphur and tryptone were omitted and starch (0.5% w/v) was added as the only carbohydrate; yeast extract concentration was 0.1% (w/v) .
  • the cell density achieved in this medium was ⁇ 10 8 cells/ml. Anaerobic conditions were achieved during the preparation of media by sparging with N 2 and following the techniques as described by Balch in Appl. Env. Microbiol. 32, 1976, pp. 781-791.
  • the culture fluid was centrifuged at 12.000 x g for 30 min. at 4°C, and the cell free supernatant was concentrated up to 100-fold using an Amicon Ultrafiltration System.
  • the cell pellet was resuspended in 50 mM sodium acetate buffer pH 5.5 and sonicated three times for 3 min. at 50% duty cycle by a BRANSON 450 sonifier.
  • the cell debris was separated from the supernatant after centrifugation at 10.000 x g for 30 min. at 4°C.
  • Temperature optima were determined by incubation of samples for 30 minutes at pH 5.5 at temperatures from 60°C to 120°C. The incubation was conducted in closed Hungate tubes in order to prevent boiling of the solution.
  • Fig. 1 shows the result (Amylase (° ) and pullulanase ( ⁇ ) ) .
  • Fig. 2 shows the result (Amylase (°) and pullulanase

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Abstract

The present invention relates to Desulfurococcus amylase and pullulanase preparations and their use in producing sweeteners and ethanol from starch.

Description

DESULFUROCOCCUS AMYLASE AND PULLULANASE
FIELD OF INVENTION
The present invention relates to a novel thermostable amylase and a novel thermostable pullulanase and their use in 5the production of sweeteners and ethanol from starch.
BACKGROUND OF THE INVENTION
The production of sweeteners from starch has been largely improved by application of different microbial enzymes to obtain better quality and yields, but the necessity of 10performing several steps of the starch-hydrolysing process at elevated temperatures means that there is still a need for new starch-hydrolysing enzymes with increased thermal stability.
It is known that Pyrococcus, e.g. Pyrococcus wosei and Pyrococcus furiosus. for reference see Arch. Microbiol. 15155. 1991, pp. 572-578, and Appl. Env. Microbiol. 56. 1990, pp.1985-1991, can produce highly thermostable amylases.
It is the object of this invention to provide an amylase and a pullulanase with temperature optimum at 80°C or above 80°C.
20SUMMARY OF THE INVENTION
We have unexpectedly found that a novel thermostable amylase and a novel thermostable pullulanase can be obtained from Desulfurococcus mucosus. a strain not previously reported to produce thermostable amylase and pullulanase; these new
25enzymes have temperature optimum around 100°C.
Accordingly, the invention provides an amylase preparation, characterized by being producible by cultivation of an amylase producing strain of Desulfurococcus mucosus. and a pullulanase preparation, characterized by being producible by
30cultivation of a pullulanase producing strain of Desulfuro¬ coccus mucosus. BRIEF DESCRIPTION OF DRAWINGS
The present invention is further illustrated by reference to the accompanying drawings, in which:
Fig. 1 shows the relative activity (% rel. ) of an amylase (°) and a pullulanase (■) of the invention at various temperatures (determined at pH 5.5 with starch and pullulan, respectively, as substrate) .
Fig. 2 shows the relative activity (% rel.) of an amylase (π) and a pullulanase (■) of the invention at various pH, determined at 90°C with starch and pullulan, respectively, as substrate.
DETAILED DISCLOSURE OF THE INVENTION
The Microorganism
According to the invention, amylase is derived from an amylase producing strain of Desulfurococcus mucosus and pullulanase is derived from a pullulanase producing strain of Desulfurococcus mucosus.
A strain representative of Desulfurococcus mucosus has been made publicly available under Accession No. DSM 2162. The number is published in the DSM Catalogue of Strains, 1993.
Production of Amylase and Pullulanase
Amylase and pullulanase of the invention may be produced by anaerobic cultivation of the above mentioned strain on a nutrient medium containing suitable carbon and nitrogen sources, such media being known in the art. Anaerobic con¬ ditions may be achieved during the preparation of media by sparging with N2 and following the anaerobic techniques as described by Balch and Wolfe in Appl. Env. Microbiol. 32, 1976, pp. 781-791. Alternatively, amylase and pullulanase of the invention can be produced by aerobic cultivation of a trans¬ formed host organism containing the appropriate genetic information from the above mentioned strain. Such transformants can be prepared and cultivated by methods known in the art.
The amylase and the pullulanase may be recovered by removing the cells from the fermentation medium (e.g. by centrifugation or filtration) and then concentrating the broth (e.g. by ultrafiltration) . If desired, the amylase and the pullulanase may be further purified by known methods.
Immunochemical Properties
The enzymes of the invention have immunochemical properties identical or partially identical (i.e. at least partially identical) to those of an enzyme derived from the strain Desulfurococcus mucosus. DSM 2162.
The immunochemical properties can be determined immunologically by cross-reaction identity tests. The identity tests can be performed by the well-known Ouchterlony double immunodiffusion procedure or by tandem crossed immunoelectro- phoresis according to Axelsen N.H. ; Handbook of Immunopre- cipitation-in-Gel Techniques; Blackwell Scientific Publications
(1983) , chapters 5 and 14. The terms "antigenic identity" and
"partial antigenic identity" are described in the same book, Chapters 5, 19 and 20.
Monospecific antisera are generated according to the above mentioned method by immunizing rabbits with the purified enzymes of the invention. The immunogens are mixed with Freund's adjuvant and injected subcutaneously into rabbits every second week. Antisera are obtained after a total im¬ munization period of 8 weeks, and immunoglobulins are prepared therefrom as described by Axelsen N.H. , supra.
The Enzymes
An amylase of the invention can be characterized by having amylase activity at temperatures of from below 60°C to approximately 120°C, having activity optimum at temperatures in the range 95-105°C, determined at pH 5.5 with starch as substrate. The amylase can also be characterized by having amylase activity at pH values of from below pH 4.0 to ap- proximately pH 11.0, having optimum in the range pH 5.5 to pH 6.5, determined at 90°C with starch as substrate.
A pullulanase of the invention can be characterized by having pullulanase activity at temperatures of from below 60°C to approximately 120°C, having activity optimum at temperatures in the range 90-105°C, determined at pH 5.5 with pullulan as substrate. The pullulanase can also be charac¬ terized by having pullulanase activity at pH values of from below pH 4.0 to approximately pH 9.0, having optimum in the range pH 4.8 to pH 5.8, determined at 90°C with pullulan as substrate.
Determination of Amylase Activity
Amylase activity is determined by measuring the amount of reducing sugar released during the incubation with starch. One unit (U) of amylase activity is defined as the amount of amylase that releases 1 μmole of reducing sugar (as maltose standard) per min. under the following assay con¬ ditions: A 0.05 ml volume of 1% soluble starch is added to 0.05 ml of 0.1 M sodium acetate buffer pH 5.5. 25 μl of enzyme solution are added to this mixture and the sample is incubated at 90°C for 30 min. The reaction is stopped by cooling on ice, and the amount of reducing sugar is determined by dinitro- salicylic acid. Sample blanks are used to correct for non- enzymatic release of reducing sugar.
Determination of Pullulanase Activity Pullulanase activity is determined by measuring the amount of reducing sugar released during the incubation with pullulan. One unit (U) of pullulanase activity is defined as the amount of pullulanase that releases 1 μmole of reducing sugar (as maltose standard) per min. under the following assay conditions: A 0.05 ml volume of 1% pullulan is added to 0.05 ml of 0.1 M sodium acetate buffer pH 5.5. 25 μl of enzyme solution are added to this mixture and the sample is incubated at 90°C for 30 min. The reaction is stopped by cooling on ice, and the amount of reducing^ sugar is determined by dinitrosalicylic acid. Sample blanks are used to correct for nonenzymatic release of reducing sugar.
Industrial Applications
The enzymes of this invention possess valuable properties allowing for various industrial applications. In particular the enzymes, in being thermostable, find potential application in the production of sweeteners and ethanol from starch. Conditions for conventional starch converting processes and liquefaction and/or saccharification processes are de¬ scribed in for instance US Patent No. 3,912,590 and EP patent publications Nos. 252,730 and 63,909.
The following example further illustrates the present invention, and it is not intended to be in any way limiting to the scope of the invention as claimed.
EXAMPLE 1
Cultivation
'The strain Desulfurococcus mucosus, DSM 2162, was recultured from glycerol-preserved cells using the medium recommended by the Deutsche Sammlung von Mikroorganismen (DSM) . The microorganisms were grown in 1 liter batch cultures under the following conditions: Medium: DSM184 (DSM184 is described in DSM Catalogue of Strains, 1993) , pH 5.8, temp. 85°C; in the medium sulphur and tryptone were omitted and starch (0.5% w/v) was added as the only carbohydrate; yeast extract concentration was 0.1% (w/v) . The cell density achieved in this medium was ≥ 108 cells/ml. Anaerobic conditions were achieved during the preparation of media by sparging with N2 and following the techniques as described by Balch in Appl. Env. Microbiol. 32, 1976, pp. 781-791.
After cultivation the culture fluid was centrifuged at 12.000 x g for 30 min. at 4°C, and the cell free supernatant was concentrated up to 100-fold using an Amicon Ultrafiltration System. The cell pellet was resuspended in 50 mM sodium acetate buffer pH 5.5 and sonicated three times for 3 min. at 50% duty cycle by a BRANSON 450 sonifier. The cell debris was separated from the supernatant after centrifugation at 10.000 x g for 30 min. at 4°C.
The following total activity (U) in both supernatant and cell extract was found: Amylase activity: 2.0 U/l Pullulanase activity: 0.8 U/l
Temperature Optima
Temperature optima were determined by incubation of samples for 30 minutes at pH 5.5 at temperatures from 60°C to 120°C. The incubation was conducted in closed Hungate tubes in order to prevent boiling of the solution.
Fig. 1 shows the result (Amylase (° ) and pullulanase (■) ) .
pH Optima
To determine pH optima, Universal buffer (Britten and
Robinson) was used to obtain values from pH 4.0 to pH 11.0.
Samples were incubated for 30 minutes at 90°C at the pH in question. Fig. 2 shows the result (Amylase (°) and pullulanase
(■) ) .

Claims

1. An amylase preparation, characterized by being producible by cultivation of an amylase producing strain of Desulfurococcus mucosus.
5 2. An amylase preparation according to claim 1, wherein said amylase producing strain is Desulfurococcus mucosus. DSM 2162.
3. An amylase according to claim 2, further charac¬ terized by:
10 (a) Activity optimum in the range pH 5.5 to pH 6.5, determined at 90°C with starch as substrate;
(b) Activity optimum at temperatures in the range 95- 105°C, determined at pH 5.5 with starch as substrate.
4. A pullulanase preparation, characterized by being 15producible by cultivation of a pullulanase producing strain of
Desulfurococcus mucosus.
5. A pullulanase preparation according to claim 4, wherein said pullulanase producing strain is Desulfurococcus mucosus, DSM 2162.
20 6. A pullulanase preparation according to claim 5, further characterized by:
(a) Activity optimum in the range pH 4.8 to pH 5.8, determined at 90°C with pullulan as substrate;
(b) Activity optimum at temperatures in the range 90- 25105°C, determined at pH 5.5 with pullulan as substrate.
7. The use of the amylase according to any of claims 1-3 in a process of producing sweeteners from starch.
8. The use of the amylase according to any of claims 1-3 in a process of producing ethanol from starch.
9. The use of the pullulanase according to any of claims 4-6 in a process of producing sweeteners from starch.
10. The use of the pullulanase according to any of claims 4-6 in a process of producing ethanol from starch.
PCT/DK1995/000098 1994-03-04 1995-03-02 Desulfurococcus amylase and pullulanase WO1995023853A1 (en)

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AU17562/95A AU1756295A (en) 1994-03-04 1995-03-02 Desulfurococcus amylase and pullulanase

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DK25694 1994-03-04

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Cited By (6)

* Cited by examiner, † Cited by third party
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EP0880590A1 (en) * 1996-02-16 1998-12-02 Recombinant Biocatalysis Inc. Esterases
WO2000001796A2 (en) * 1998-07-02 2000-01-13 Novozymes A/S Starch debranching enzymes
US6043074A (en) * 1999-05-07 2000-03-28 Novo Nordisk A/S Desulfurococcus amylopullulanase
US6265197B1 (en) 1998-07-02 2001-07-24 Novozymes A/S Krogshoejvej Starch debranching enzymes
US7288400B2 (en) 1996-02-16 2007-10-30 Verenium Corporation Nucleic acids encoding esterases and methods of making and using them
WO2011058105A1 (en) 2009-11-13 2011-05-19 Novozymes A/S A brewing method

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US7288400B2 (en) 1996-02-16 2007-10-30 Verenium Corporation Nucleic acids encoding esterases and methods of making and using them
EP1550721A3 (en) * 1996-02-16 2007-04-18 Diversa Corporation Esterases
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US7374922B2 (en) 1998-07-02 2008-05-20 Novozymes A/S Starch debranching enzymes
US6265197B1 (en) 1998-07-02 2001-07-24 Novozymes A/S Krogshoejvej Starch debranching enzymes
WO2000001796A3 (en) * 1998-07-02 2000-03-09 Novo Nordisk As Starch debranching enzymes
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US6043074A (en) * 1999-05-07 2000-03-28 Novo Nordisk A/S Desulfurococcus amylopullulanase
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