EP0793716A1 - AMYLASE ET PULLULANASE DE $i(THERMOCOCCUS) - Google Patents

AMYLASE ET PULLULANASE DE $i(THERMOCOCCUS)

Info

Publication number
EP0793716A1
EP0793716A1 EP95910468A EP95910468A EP0793716A1 EP 0793716 A1 EP0793716 A1 EP 0793716A1 EP 95910468 A EP95910468 A EP 95910468A EP 95910468 A EP95910468 A EP 95910468A EP 0793716 A1 EP0793716 A1 EP 0793716A1
Authority
EP
European Patent Office
Prior art keywords
pullulanase
amylase
starch
thermococcus
determined
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Withdrawn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
EP95910468A
Other languages
German (de)
English (en)
Inventor
Carsten Sjoholm
G. Technische Uni. 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 EP0793716A1 publication Critical patent/EP0793716A1/fr
Withdrawn legal-status Critical Current

Links

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
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • 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
    • 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
    • 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
    • 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)

Definitions

  • the present invention relates to a novel thermostable amylase and a novel thermostable pullulanase and their use in 5 the 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 10 performing 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 the genus Thermococcus. a genus not previously reported to produce thermostable amylase and pullulanase; these new enzymes
  • the invention provides an amylase preparation, characterized by being producible by cultivation of an amylase producing strain of the genus Thermococcus. 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 the genus Thermococcus, in particular Thermococcus celer.
  • pullulanase is derived from a pullulanase producing strain of the genus Thermococcus, in particular Thermococcus celer.
  • a strain representative of Thermococcus celer has been made publicly available under Accession No. DSM 2476. 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
  • 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 Thermococcus celer, DSM 2476.
  • 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- cipitatio ⁇ -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.
  • 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 85-95°C, determined at pH 5.5 with starch as sub- strate.
  • the amylase can also be characterized by having amylase activity at pH values of from below pH 4.5 to approximately pH 9.0, having optimum in the range pH 5.0 to pH 6.0, 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 above 120°C, having activity optimum at temperatures in the range 85-95°C, determined at pH 5.5 with pullulan as substrate.
  • the pullulanase can also be characterized by having pullulanase activity at pH values of from below pH 4.5 to approximately pH 9.8, having optimum in the range pH 5.0 to pH 6.0, 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.
  • the strain Thermococcus celer, DSM 2476 was re- cultured from glycerol-preserved cells using the medium recommended by the Deutsche Sammlung von Mikroorganismen (DSM) .
  • the microorganisms were grown in 1 liter of batch cultures under the following conditions: Medium: DSM266 (DSM266 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.
  • Temperature optima were determined by incubation of samples for 30 minutes at pH 5.5 at temperatures from 60°C to
  • Fig. 1 shows the result (Amylase (°) and pullulanase
  • Fig. 2 shows the result (Amylase (°) and pullulanase ( ⁇ ) ) .

Landscapes

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

Abstract

L'invention concerne des préparations d'amylase et de pullulanase de Thermococcus et leur utilisation pour produire 5 édulcorants et de l'éthanol à partir de l'amidon. En particulier, ces enzymes sont dérivées du Thermococcus celer.
EP95910468A 1994-03-04 1995-03-02 AMYLASE ET PULLULANASE DE $i(THERMOCOCCUS) Withdrawn EP0793716A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DK25594 1994-03-04
DK255/94 1994-03-04
PCT/DK1995/000097 WO1995023852A1 (fr) 1994-03-04 1995-03-02 AMYLASE ET PULLULANASE DE $i(THERMOCOCCUS)

Publications (1)

Publication Number Publication Date
EP0793716A1 true EP0793716A1 (fr) 1997-09-10

Family

ID=8091485

Family Applications (1)

Application Number Title Priority Date Filing Date
EP95910468A Withdrawn EP0793716A1 (fr) 1994-03-04 1995-03-02 AMYLASE ET PULLULANASE DE $i(THERMOCOCCUS)

Country Status (3)

Country Link
EP (1) EP0793716A1 (fr)
AU (1) AU1756195A (fr)
WO (1) WO1995023852A1 (fr)

Families Citing this family (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5763234A (en) * 1996-07-25 1998-06-09 Florida Atlantic University Secosteroids, and method for producing same
WO1998026058A1 (fr) * 1996-12-11 1998-06-18 Universite De Reims Champagne Ardennes Alpha-glucosidase et pullulanase thermostables et leurs utilisations
FR2763598B1 (fr) * 1997-03-12 2001-07-06 Univ Reims Champagne Ardennes Nouvelle pullalanase thermostable et son utilisation industrielle
FR2756844B1 (fr) * 1996-12-11 1999-02-12 Univ Reims Champagne Ardennes Nouvelle alpha-glucosidase et nouvelle pullulanase thermostables et leurs utilisations industrielles
FR2778412B1 (fr) * 1998-05-05 2002-08-09 Univ Reims Champagne Ardennes Procede pour preparer une enzyme alpha-amylase thermophile et enzyme ainsi obtenue
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
BR112012011106C8 (pt) 2009-11-13 2019-11-26 Novozymes As método de mosturação
WO2011076123A1 (fr) 2009-12-22 2011-06-30 Novozymes A/S Compositions comprenant un polypeptide renforçateur et un enzyme dégradant l'amidon, et utilisations correspondantes

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5023094A (en) * 1989-08-10 1991-06-11 Gist-Brocades N.V. Retarding the firming of bread crumb during storage

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO9523852A1 *

Also Published As

Publication number Publication date
WO1995023852A1 (fr) 1995-09-08
AU1756195A (en) 1995-09-18

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