WO1995023852A1 - AMYLASE ET PULLULANASE DE $i(THERMOCOCCUS) - Google Patents

AMYLASE ET PULLULANASE DE $i(THERMOCOCCUS) Download PDF

Info

Publication number
WO1995023852A1
WO1995023852A1 PCT/DK1995/000097 DK9500097W WO9523852A1 WO 1995023852 A1 WO1995023852 A1 WO 1995023852A1 DK 9500097 W DK9500097 W DK 9500097W WO 9523852 A1 WO9523852 A1 WO 9523852A1
Authority
WO
WIPO (PCT)
Prior art keywords
pullulanase
amylase
starch
thermococcus
determined
Prior art date
Application number
PCT/DK1995/000097
Other languages
English (en)
Inventor
Carsten SJØHOLM
Garabed Antranikian
Original Assignee
Novo Nordisk A/S
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 A/S filed Critical Novo Nordisk A/S
Priority to AU17561/95A priority Critical patent/AU1756195A/en
Priority to EP95910468A priority patent/EP0793716A1/fr
Publication of WO1995023852A1 publication Critical patent/WO1995023852A1/fr

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 ( ⁇ ) ) .

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.
PCT/DK1995/000097 1994-03-04 1995-03-02 AMYLASE ET PULLULANASE DE $i(THERMOCOCCUS) WO1995023852A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU17561/95A AU1756195A (en) 1994-03-04 1995-03-02 (thermococcus) amylase and pullulanase
EP95910468A EP0793716A1 (fr) 1994-03-04 1995-03-02 AMYLASE ET PULLULANASE DE $i(THERMOCOCCUS)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK255/94 1994-03-04
DK25594 1994-03-04

Publications (1)

Publication Number Publication Date
WO1995023852A1 true WO1995023852A1 (fr) 1995-09-08

Family

ID=8091485

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DK1995/000097 WO1995023852A1 (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)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998004676A2 (fr) * 1996-07-25 1998-02-05 Florida Atlantic University Research Corporation Procede de production de composes de derives marins et composes ainsi produits
FR2756844A1 (fr) * 1996-12-11 1998-06-12 Univ Reims Champagne Ardennes Nouvelle alpha-glucosidase et nouvelle pullulanase thermostables et leurs utilisations industrielles
WO1998026058A1 (fr) * 1996-12-11 1998-06-18 Universite De Reims Champagne Ardennes Alpha-glucosidase et pullulanase thermostables et leurs utilisations
FR2763598A1 (fr) * 1997-03-12 1998-11-27 Univ Reims Champagne Ardennes Nouvelle pullalanase thermostable et son utilisation industrielle
FR2778412A1 (fr) * 1998-05-05 1999-11-12 Univ Reims Champagne Ardennes Procede pour preparer une enzyme alpha-amylase thermophile et enzyme ainsi obtenue
WO2000001796A2 (fr) * 1998-07-02 2000-01-13 Novozymes A/S Enzymes de deramification de l'amidon
US6265197B1 (en) 1998-07-02 2001-07-24 Novozymes A/S Krogshoejvej Starch debranching enzymes
WO2011058105A1 (fr) 2009-11-13 2011-05-19 Novozymes A/S Procédé de brassage
WO2011087836A2 (fr) 2009-12-22 2011-07-21 Novozymes A/S Variants de pullulanase et utilisations de ceux-ci

Citations (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

Patent Citations (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 (5)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, FILE 357, DERWENT BIOTECHNOLOGY ABS, Dialog Accession No. 163760, DBA Accession No. 94-06311, ZEIKUS J.G. et al., "Thermozymes: from Nature to the Biodesign of Industrial Saccharidases - Thermoanaerobacterium sp. Thermoanaerobacterium sp., Thermoanaerobacter sp., Thermotage sp., *
DIALOG INFORMATION SERVICES, FILE 55, BIOSIS PREVIEWS, Dialog Accession No. 10504212, BIOSIS No. 96104212, BROWN S.H. et al., "Characterization of Amylolytic Enzymes Having Both Alpha-1 4 and Alpha-1 6 Hydrolytic Activity from the Thermophilic Archaea Pyrococcus-furiosus and Thermococcus-litoralis"; & APPL. ENVIRON. *
DIALOG INFORMATION SERVICES, FILE 55, BIOSIS PREVIEWS, Dialog Accession No. 11491261, BIOSIS No. 98091261, CANGANELLA F. et al., "Characterization of Amylolytic and Pullulytic Enzymes from Thermophilic Archaea and from a New Fervidobacterium Species"; & APPLIED MICROBIOLOGY AND BIOTECHNOLOGY, 42 (2-3), 1994, 239-245. *
NATIONAL LIBRARY OF MEDICINE, (NLM), MEDLINE, Medline Accession No. 94079331, ADAMS M.W., "Enzymes and Proteins from Organisms That Grow Near and Above 100 Degrees C"; & ANNU. REV. MICROBIOL., 1993, 47:627-58. *
TIBTECH, Volume 7, Sept. 1989, BADAL C. SAHA et al., "Reviews, Novel Highly Thermostable Pullulanase from Thermophiles", pages 234. *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6046041A (en) * 1996-07-25 2000-04-04 Univ Florida Atlantic Method for producing marine-derived compounds and novel compounds produced therefrom
WO1998004676A3 (fr) * 1996-07-25 1998-05-28 Univ Florida Atlantic Procede de production de composes de derives marins et composes ainsi produits
WO1998004676A2 (fr) * 1996-07-25 1998-02-05 Florida Atlantic University Research Corporation Procede de production de composes de derives marins et composes ainsi produits
FR2756844A1 (fr) * 1996-12-11 1998-06-12 Univ Reims Champagne Ardennes Nouvelle alpha-glucosidase et nouvelle pullulanase thermostables et leurs utilisations industrielles
WO1998026058A1 (fr) * 1996-12-11 1998-06-18 Universite De Reims Champagne Ardennes Alpha-glucosidase et pullulanase thermostables et leurs utilisations
FR2763598A1 (fr) * 1997-03-12 1998-11-27 Univ Reims Champagne Ardennes Nouvelle pullalanase thermostable et son utilisation industrielle
FR2778412A1 (fr) * 1998-05-05 1999-11-12 Univ Reims Champagne Ardennes Procede pour preparer une enzyme alpha-amylase thermophile et enzyme ainsi obtenue
WO2000001796A2 (fr) * 1998-07-02 2000-01-13 Novozymes A/S Enzymes de deramification de l'amidon
WO2000001796A3 (fr) * 1998-07-02 2000-03-09 Novo Nordisk As Enzymes de deramification de l'amidon
US6265197B1 (en) 1998-07-02 2001-07-24 Novozymes A/S Krogshoejvej Starch debranching enzymes
US7374922B2 (en) 1998-07-02 2008-05-20 Novozymes A/S Starch debranching enzymes
EP2216401A1 (fr) * 1998-07-02 2010-08-11 Novozymes A/S Enzymes de déramification de l'amidon
US7816113B2 (en) 1998-07-02 2010-10-19 Novozymes Als Starch debranching enzymes
WO2011058105A1 (fr) 2009-11-13 2011-05-19 Novozymes A/S Procédé de brassage
WO2011087836A2 (fr) 2009-12-22 2011-07-21 Novozymes A/S Variants de pullulanase et utilisations de ceux-ci
US8703465B2 (en) 2009-12-22 2014-04-22 Novozymes A/S Pullulanase variants and uses thereof

Also Published As

Publication number Publication date
AU1756195A (en) 1995-09-18
EP0793716A1 (fr) 1997-09-10

Similar Documents

Publication Publication Date Title
US4560651A (en) Debranching enzyme product, preparation and use thereof
US4536477A (en) Thermostable glucoamylase and method for its production
CA1214407A (fr) Amylase maltogenique, preparation et utilisation
EP0063909B1 (fr) Produit d'enzyme débranchant, sa préparation et utilisation
US4284722A (en) Heat and acid-stable alpha-amylase enzymes and processes for producing the same
CA1142464A (fr) Systeme de culture thermophile anaerobie
CA2088592A1 (fr) Pullulanases thermostables
KR890001827B1 (ko) α-아밀라제의제법
US5688668A (en) Pyrodictium xylanase amylase and pullulanase
EP0195802B1 (fr) G(b) -amylase thermostable
WO1995023852A1 (fr) AMYLASE ET PULLULANASE DE $i(THERMOCOCCUS)
JPH0330672A (ja) 新規耐熱性および耐酸性プルラナーゼ酵素およびその製造方法
EP0753056A1 (fr) Amylase et pullulanase de desulfurococcus
US4613570A (en) Novel thermostable, aciduric alpha-amylase and method for its production
US5714369A (en) Fervidobacterium amylase and pullulanase
CA1081633A (fr) Production d'alpha-amylases resistant a la chaleur et aux acides
US5281527A (en) Process for producing pullalanase
US5882906A (en) Staphylothermus amylase
CA2094398A1 (fr) Enzyme d'isomerisation
WIMMER et al. A novel type of thermostable α-D-glucosidase from Thermoanaerobacter thermohydrosulfuricus exhibiting maltodextrinohydrolase activity
JP7460978B2 (ja) β－グルコシダーゼを生産する微生物及び該β－グルコシダーゼを生産する微生物を用いたセルロース系バイオマスの糖化方法。
JP3557276B2 (ja) 酵素をコードするｄｎａとそれを含む組換えｄｎａ並びに形質転換体
CN116064480A (zh) 一种热稳定性提高的α-葡萄糖苷酶环化突变体及其构建方法
JPS6331194B2 (fr)
JPS6331195B2 (fr)

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK EE ES FI GB GE HU JP KE KG KP KR KZ LK LR LT LU LV MD MG MN MW MX NL NO NZ PL PT RO RU SD SE SG SI SK TJ TT UA UG US UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application
WWE Wipo information: entry into national phase

Ref document number: 1995910468

Country of ref document: EP

ENP Entry into the national phase

Ref country code: US

Ref document number: 1996 704637

Date of ref document: 19960909

Kind code of ref document: A

Format of ref document f/p: F

NENP Non-entry into the national phase

Ref country code: CA

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

WWP Wipo information: published in national office

Ref document number: 1995910468

Country of ref document: EP

WWW Wipo information: withdrawn in national office

Ref document number: 1995910468

Country of ref document: EP