WO1998022613A1 - Utilisation d'un domaine de fixation sur des glucides dans le traitement de l'amidon - Google Patents

Utilisation d'un domaine de fixation sur des glucides dans le traitement de l'amidon Download PDF

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Publication number
WO1998022613A1
WO1998022613A1 PCT/DK1997/000537 DK9700537W WO9822613A1 WO 1998022613 A1 WO1998022613 A1 WO 1998022613A1 DK 9700537 W DK9700537 W DK 9700537W WO 9822613 A1 WO9822613 A1 WO 9822613A1
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WO
WIPO (PCT)
Prior art keywords
cbd
starch
enzyme
xylanase
carbohydrate
Prior art date
Application number
PCT/DK1997/000537
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English (en)
Inventor
Sven Pedersen
Claus Christophersen
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 AU49419/97A priority Critical patent/AU4941997A/en
Priority to EP97912081A priority patent/EP0941359A1/fr
Publication of WO1998022613A1 publication Critical patent/WO1998022613A1/fr

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    • 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

Definitions

  • the present invention relates, inter alia, to the use of a combination of a carbohydrate-binding domain ("CBD”) and an enzyme of a type employed in industrial starch processing [notably starch processing for the production (vide infra) of sweeteners, particularly glucose- and/or fructose-containing syrups] , especially an amylolytic enzyme, such as an ⁇ -amylase employed in a so-called “starch liquefaction" process (vide infra) in which starch is degraded (often termed “dextrinized”) to smaller oligo- and/or polysaccharide fragments, or a debranching enzyme (such as an isoamylase or a pullulanase) employed to debranch amylopectin-derived starch fragments in connection with the so-called “saccharification” process (vide infra) which is normally carried out after the liquefaction stage.
  • CBD carbohydrate-binding domain
  • the present invention is, inter alia , of value in the field of starch processing (starch conversion) .
  • starch conversion starch conversion
  • Conditions for conventional starch conversion processes and for liquefaction and/or saccharification processes are described in, e.g., US 3,912,590 and in EP 0 252 730 and EP 0 063 909.
  • a "traditional" process for the production of glucose- and fructose-containing syrups from starch normally consists of three consecutive enzymatic processes, viz. a liquefaction process followed by a sacchari- fication process and (for production of fructose-containing syrups) an isomerization process.
  • starch initially in the form of a starch suspension in aqueous medium
  • dextrins oligo- and polysaccharide fragments of starch
  • TermamylTM (Bacillus licheniformis ⁇ -amylase) , available from Novo Nordisk A/S, Bagsvaerd, Denmark], typically at pH values between 5.5 and 6.2 and at temperatures of 95-160°C for a period of approximately 2 hours.
  • approximately 1 mM of calcium (ca. 40 ppm free calcium ions) is typically added to the starch suspension.
  • the dextrins are converted into dextrose (D-glucose) by addition of a glucoamylase (amyloglucosidase, EC 3.2.1.3; e.g.
  • One aspect of the invention relates to an improved enzymatic process for liquefying starch employing a combination of a carbohydrate-binding domain (CBD; vide infra) and at least one liquefying amylolytic enzyme, such as an ⁇ -amylase.
  • CBD carbohydrate-binding domain
  • Example 2 a pure CBD di-mer derived from Clostridium stercorarium (NCIMB 11754) XynA (GenBank and SWISS-PROT Accession No.13325) is used in combination with a debranching enzyme (Promozyme TM) for debranchi»ng amylopecti ⁇ n.
  • the pure CBD may be provided using techniques well-known in the art, e.g. as described by Ong E. et al. (1993) , Biotechnology and
  • the dispersion with the small gluten agglomerates and starch is pumped to a set of hydrocyclones, where a centrifugal separation takes place.
  • the gluten and the "B"-starch being the lightest fraction leaves the top of the hydrocyclones together and the gluten is separated from the "B"-starch by screens.
  • the underflow from the hydrocyclones consists mainly of "A"-starch, while pentosan (or fibres) are found in both fractions.
  • the fractions are further cleaned by a series of washing/concentration steps.
  • xylanase As defined herein and a cellulase
  • a CBD in combination with a xylanase may also be used for reducing the viscosity of plant material.
  • the viscosity reduction may be important, e.g. in a continuous wheat separation process, in that an increased wheat flour flow may be obtained. Furthermore, the viscosity reduction is important in the preparation of food or feed and in brewing, cf Visser et al . , Xylans and Xylanases, (1991).
  • CBDs are found as integral parts of large polypeptides or proteins consisting of two or more polypeptide amino acid sequence regions, especially in hydrolytic enzymes (hydrolases) which typically comprise a catalytic domain containing the active site for substrate hydrolysis and a carbohydrate-binding domain (CBD) for binding to the carbohydrate substrate in question.
  • hydrolytic enzymes hydrolytic enzymes
  • CBDs carbohydrate-binding domain
  • Such enzymes can comprise more than one catalytic domain and one, two or three CBDs, and they may further comprise one or more polypeptide amino acid sequence regions linking the CBD(s) with the catalytic domain (s) , a region of the latter type usually being denoted a "linker".
  • That part of a polypeptide or protein (e.g. hydrolytic enzyme) which constitutes a CBD per se typically consists of more than about 30 and less than about 250 amino acid residues.
  • those CBDs listed and classified in Family I in accordance with P. Tomme et al. (op . cit . ) consist of 33-37 amino acid residues
  • those listed and classified in Family Ila consist of 95-108 amino acid residues
  • those listed and classified in Family VI consist of 85-92 amino acid residues
  • one CBD derived from a cellulase from Clostridium thermocellum listed and classified in Family VII consists of 240 amino acid residues.
  • the molecular weight of an amino acid sequence constituting a CBD per se will typically be in the range of from about 4kD to about 40kD, and usually below about 35kD.
  • a preferred useful acid cellulase is one derived from or producible by fungi from the group of species consisting of Trichoderma viride, Trichoderma reesei, Trichoderma longibrachiatum, Myrothecium verrucaria, Aspergillus niger, Aspergillus oryzae, Phanaerochaete chrysosporium, Neurospora crassa, Neocallimastix partriciarum and Botrytis cinerea .
  • a preferred cellulase is an alkaline endoglucanase which is immunologically reactive with an antibody raised against a highly purified " 43kD endoglucanase derived from Humicola in ⁇ olen ⁇ DSM 1800, or which is a derivative of the latter ⁇ 43kD endoglucanase and exhibits cellulase activity (e.g. CarezymeTM) .
  • a cellulose-binding domain of, e.g., a cellulase several genetic engineering approaches may be used.
  • One method uses restriction enzymes to remove a portion of the gene and then to fuse the remaining gene-vector fragment in frame to obtain a mutated gene that encodes a protein truncated for a particular gene fragment.
  • Another method involves the use of exonucleases such as Bal31 to systematically delete nucleotides either externally from the 5' and the 3' ends of the DNA or internally from a restricted gap within the gene.
  • exonucleases such as Bal31 to systematically delete nucleotides either externally from the 5' and the 3' ends of the DNA or internally from a restricted gap within the gene.
  • substrate-binding e.g. cellulose-binding
  • Appropriate substrates for evaluating the binding ability include cellulosic materials such as AvicelTM and cotton fibres.
  • Amylolytic enzymes at least in the context of the present invention enzymes within the group of enzymes classified under EC 3.2.1 (e.g. pullulanase) and EC 2.4.1. (e.g. D-enzyme and Q-enzyme) .
  • Amylases (in particular ⁇ -amylases) which are appropriate for use in combination with CBDs in the context of the present invention include those of bacterial or fungal origin. Chemically or genetically modified mutants of such amylases are included in this connection.
  • Relevant ⁇ -amylases include, for example, ⁇ - amylases obtainable from Bacillu ⁇ species, in particular a special strain of B. licheniformis, described in more detail in GB 1296839.
  • Relevant commercially available amylases include
  • DuramylTM, TermamylTM, FungamylTM and BANTM all available from Novo Nordisk A/S, Bagsvaerd, Denmark
  • RapidaseTM and Maxamyl PTM available from Gist-Brocades, Holland
  • Isoamylases (EC 3.2.1.68) appropriate for use in combination with CBDs in the context of the present invention include those of bacterial origin. Chemically or genetically mod- ified mutants of such isoamylases are included in this connection.
  • Relevant isoamylases include, for example, isoamylases obtainable from P ⁇ eudomona ⁇ species, (e.g- P ⁇ eudomona ⁇ sp. SMP1 or P. amyloderomo ⁇ a SB15) , Bacillu ⁇ species (e.g. B. amyloliquefacien ⁇ ) , Flavobacterium species or Cytophaga (Lysojba ⁇ ter) species.
  • Pullulanases EC 3.2.1.41
  • pullulanases appropriate for use in combination with CBDs in the context of the present invention include those of bacterial origin. Chemically or genetically mod- ified mutants of such pullulanases are included in this connection.
  • Relevant pullulanases include, for example, pullulanases obtainable from Bacillu ⁇ species (e.g. B. acidopullulyticu ⁇ ; such as PromozymeTM, from Novo Nordisk A/S) .
  • a Q-enzyme may e.g. be derived from a strain of Bacillus sp. such as B. megaterium or B. stearothermophilus or other branching enzymes described in EP 418,945.
  • thermophilu ⁇ 25 thermophilu ⁇ , Thermococcu ⁇ lithorali ⁇ , Clo ⁇ tridium butyricum , Streptococcu ⁇ pneumoniae , E . coli or from Solanum tuberosum (potato) .
  • CBD e.g. selected among those types of CBDs mentioned herein
  • more than one CBD may be used in
  • CBD Cellulose-binding Domain di-mer derived from Clostridium ⁇ tercorarium (NCIMB 11754) XynA (GenBank and SWISS-PROT Accession No.13325 or Sakka et al., (1993), Biosci. Biotechnol. Biochem. 57 (2), p. 273-277. "Nucleotide sequence of the Clostridium ⁇ tercorarium xynA gene encoding xylanase A: identification of catalytic and Cellulose-binding domains or Sakka et al. (1996), Ann. N. Y. Acad. Sci. 782, p. 241-251, "Identification and characterization of Cellulose-binding domains in xylanase A of Clo ⁇ tridium ⁇ tercorarium) . Amylopectin (Waxy maize starch, Cerestar) Flour
  • PUN Pullulanase Unit Novo
  • the endo-xylanase activity is determined by an assay, in which the xylanase sample is incubated with a remazol-xylan substrate (4-O-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue R, Fluka) , pH 6.0. The incubation is performed at 50°C for 30 min. The background of non-degraded dyed substrate is precipitated by ethanol. The remaining blue colour in the supernatant is determined spectrophotometrically at 585 n and is proportional to the endoxylanase activity.
  • a remazol-xylan substrate (4-O-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue R, Fluka) , pH 6.0.
  • the incubation is performed at 50°C for 30 min.
  • the background of non-degraded dyed substrate is precipitated by ethanol.
  • the remaining blue colour in the supernatant is determined
  • CBDs from Clo ⁇ tridium ⁇ teacorarium enhance the debranching efficiency of debranching enzyme.

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  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Microbiology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
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  • Genetics & Genomics (AREA)
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Abstract

L'invention concerne un procédé de liquéfaction d'amidon consistant à traiter un substrat d'amidon, dans un milieu aqueux, avec un domaine de fixation sur des glucides, ainsi qu'avec au moins une enzyme amylolytique.
PCT/DK1997/000537 1996-11-21 1997-11-21 Utilisation d'un domaine de fixation sur des glucides dans le traitement de l'amidon WO1998022613A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
AU49419/97A AU4941997A (en) 1996-11-21 1997-11-21 Use of carbohydrate-binding domain in starch processing
EP97912081A EP0941359A1 (fr) 1996-11-21 1997-11-21 Utilisation d'un domaine de fixation sur des glucides dans le traitement de l'amidon

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK1331/96 1996-11-21
DK133196 1996-11-21

Publications (1)

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WO1998022613A1 true WO1998022613A1 (fr) 1998-05-28

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AU (1) AU4941997A (fr)
WO (1) WO1998022613A1 (fr)

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000910A2 (fr) * 2000-06-23 2002-01-03 Novozymes A/S Procede de trempage
FR2874930A1 (fr) * 2004-09-03 2006-03-10 Df3 Sarl Sarl Procede d'obtention de fractions issues du son de cereales ainsi que les fractions ainsi obtenues
US7129069B2 (en) 2003-10-28 2006-10-31 Novo Zymes Als Hybrid enzymes
US7883883B2 (en) 2003-06-25 2011-02-08 Novozymes A/S Enzymes for starch processing
WO2011049945A2 (fr) 2009-10-23 2011-04-28 Danisco Us Inc. Procédés destinés à réduire le saccharide donnant une couleur bleue
US8323945B2 (en) 2008-06-06 2012-12-04 Danisco Us Inc. Variant alpha-amylases from Bacillus subtilis and methods of uses, thereof
US8753859B2 (en) 2009-05-19 2014-06-17 Dupont Nutrition Biosciences Aps Amylase polypeptides
US9040278B2 (en) 2008-06-06 2015-05-26 Danisco Us Inc. Production of glucose from starch using alpha-amylases from Bacillus subtilis
US9040279B2 (en) 2008-06-06 2015-05-26 Danisco Us Inc. Saccharification enzyme composition and method of saccharification thereof
US9044544B2 (en) 2008-11-21 2015-06-02 Baxter International Inc. Dialysis machine having auto-connection system with roller occluder
US9303254B2 (en) 2008-04-30 2016-04-05 Danisco Us Inc. Chimeric alpha-amylase variants

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2312559A1 (fr) * 1975-05-30 1976-12-24 Baxter Laboratories Inc Procede de maltage de l'orge
US4746517A (en) * 1985-12-03 1988-05-24 Gist-Brocades S.A. Production of beer
US4914029A (en) * 1987-11-17 1990-04-03 Dorr-Oliver Incorporated Process for steeping cereals with a new enzyme preparation
US5023176A (en) * 1985-12-03 1991-06-11 Gist-Brocades N.V. Production of glucose syrups and purified starches from wheat and other cereal starches containing pentosans

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2312559A1 (fr) * 1975-05-30 1976-12-24 Baxter Laboratories Inc Procede de maltage de l'orge
US4746517A (en) * 1985-12-03 1988-05-24 Gist-Brocades S.A. Production of beer
US5023176A (en) * 1985-12-03 1991-06-11 Gist-Brocades N.V. Production of glucose syrups and purified starches from wheat and other cereal starches containing pentosans
US4914029A (en) * 1987-11-17 1990-04-03 Dorr-Oliver Incorporated Process for steeping cereals with a new enzyme preparation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
FILE WPI, Derwent Accession No. 72-43376T, FERMENTATION PRODUCTS RES: "Ethanol Continuous Mfr - from Starchy Raw Materials Saccharification with Alpha-Amylase, Glucoamylyse, Cellulase and Hemicellulase to Imp"; & SU,A,316 719, DW7227. *
FILE WPI, Derwent Accession No. 82-18787E, HANKYU KYOEI BUSSAN KK: "Saccharification of Starch without Steaming, for Use as Fuel - by Sterilising Starch with Addn. of Acid, Grinding and Saccharifying with (Hemi) Cellulase, Pectinase, Glucomylase and Acidic Alpha-Amylase"; & JP,A,57 018 991, (30-01-82), DW8210. *
FILE WPI, Derwent Accession No. 89-238844, ASAIS: "Sweet Potato Syrup Prodn. -by Slicing Peeled Potatoes, Desizing in Boiling Water, Steaming, Treating with Alpha-Amylase, etc"; & JP,A,01 174 394, (10-07-89), DW8933. *

Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002000910A2 (fr) * 2000-06-23 2002-01-03 Novozymes A/S Procede de trempage
WO2002000910A3 (fr) * 2000-06-23 2002-05-10 Novozymes As Procede de trempage
US7883883B2 (en) 2003-06-25 2011-02-08 Novozymes A/S Enzymes for starch processing
US8263381B2 (en) 2003-06-25 2012-09-11 Novozyms A/S Enzymes for starch processing
US7129069B2 (en) 2003-10-28 2006-10-31 Novo Zymes Als Hybrid enzymes
US7312055B2 (en) 2003-10-28 2007-12-25 Novozymes A/S Hybrid enzymes
US7749744B2 (en) 2003-10-28 2010-07-06 Novozymes A/S Hybrid enzymes
FR2874930A1 (fr) * 2004-09-03 2006-03-10 Df3 Sarl Sarl Procede d'obtention de fractions issues du son de cereales ainsi que les fractions ainsi obtenues
WO2006027529A3 (fr) * 2004-09-03 2006-05-04 Df3 Procede d'obtention de fractions issues du son de cereales ainsi que les fractions ainsi obtenues
US9303254B2 (en) 2008-04-30 2016-04-05 Danisco Us Inc. Chimeric alpha-amylase variants
US8975056B2 (en) 2008-06-06 2015-03-10 Danisco Us Inc. Variant alpha-amylases from Bacillus subtilis and methods of uses, thereof
US8323945B2 (en) 2008-06-06 2012-12-04 Danisco Us Inc. Variant alpha-amylases from Bacillus subtilis and methods of uses, thereof
US9040278B2 (en) 2008-06-06 2015-05-26 Danisco Us Inc. Production of glucose from starch using alpha-amylases from Bacillus subtilis
US9040279B2 (en) 2008-06-06 2015-05-26 Danisco Us Inc. Saccharification enzyme composition and method of saccharification thereof
US9090887B2 (en) 2008-06-06 2015-07-28 Danisco Us Inc. Variant alpha-amylases from Bacillus subtilis and methods of use, thereof
US9044544B2 (en) 2008-11-21 2015-06-02 Baxter International Inc. Dialysis machine having auto-connection system with roller occluder
US8753859B2 (en) 2009-05-19 2014-06-17 Dupont Nutrition Biosciences Aps Amylase polypeptides
WO2011049945A2 (fr) 2009-10-23 2011-04-28 Danisco Us Inc. Procédés destinés à réduire le saccharide donnant une couleur bleue

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Publication number Publication date
AU4941997A (en) 1998-06-10
EP0941359A1 (fr) 1999-09-15

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