EP0753056A1 - Amylase et pullulanase de desulfurococcus - Google Patents

Amylase et pullulanase de desulfurococcus

Info

Publication number
EP0753056A1
EP0753056A1 EP95910469A EP95910469A EP0753056A1 EP 0753056 A1 EP0753056 A1 EP 0753056A1 EP 95910469 A EP95910469 A EP 95910469A EP 95910469 A EP95910469 A EP 95910469A EP 0753056 A1 EP0753056 A1 EP 0753056A1
Authority
EP
European Patent Office
Prior art keywords
pullulanase
amylase
starch
determined
desulfurococcus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95910469A
Other languages
German (de)
English (en)
Inventor
Carsten Novo Nordisk A/S SJOHOLM
G. Technische Univ. Hamburg-Harburg ANTRANIKIAN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Original Assignee
Novo Nordisk AS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk AS filed Critical Novo Nordisk AS
Publication of EP0753056A1 publication Critical patent/EP0753056A1/fr
Withdrawn legal-status Critical Current

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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)
    • C12N9/24—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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  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne des préparations d'amylase et de pullulanase de desulfurococcus et leur utilisation pour produire des édulcorants et de l'éthanol à partir de l'amidon.
EP95910469A 1994-03-04 1995-03-02 Amylase et pullulanase de desulfurococcus Withdrawn EP0753056A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DK256/94 1994-03-04
DK25694 1994-03-04
PCT/DK1995/000098 WO1995023853A1 (fr) 1994-03-04 1995-03-02 Amylase et pullulanase de desulfurococcus

Publications (1)

Publication Number Publication Date
EP0753056A1 true EP0753056A1 (fr) 1997-01-15

Family

ID=8091496

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95910469A Withdrawn EP0753056A1 (fr) 1994-03-04 1995-03-02 Amylase et pullulanase de desulfurococcus

Country Status (3)

Country Link
EP (1) EP0753056A1 (fr)
AU (1) AU1756295A (fr)
WO (1) WO1995023853A1 (fr)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7288400B2 (en) 1996-02-16 2007-10-30 Verenium Corporation Nucleic acids encoding esterases and methods of making and using them
US5942430A (en) * 1996-02-16 1999-08-24 Diversa Corporation Esterases
DE69939829D1 (de) * 1998-07-02 2008-12-11 Novozymes As Stärke "debranching-enzyme"
US6265197B1 (en) 1998-07-02 2001-07-24 Novozymes A/S Krogshoejvej Starch debranching enzymes
US6043074A (en) * 1999-05-07 2000-03-28 Novo Nordisk A/S Desulfurococcus amylopullulanase
BR112012011106C8 (pt) 2009-11-13 2019-11-26 Novozymes As método de mosturação

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9523853A1 *

Also Published As

Publication number Publication date
AU1756295A (en) 1995-09-18
WO1995023853A1 (fr) 1995-09-08

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