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EP0941359A1 - 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

Info

Publication number
EP0941359A1
EP0941359A1 EP97912081A EP97912081A EP0941359A1 EP 0941359 A1 EP0941359 A1 EP 0941359A1 EP 97912081 A EP97912081 A EP 97912081A EP 97912081 A EP97912081 A EP 97912081A EP 0941359 A1 EP0941359 A1 EP 0941359A1
Authority
EP
European Patent Office
Prior art keywords
cbd
starch
enzyme
xylanase
carbohydrate
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Withdrawn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
EP97912081A
Other languages
German (de)
English (en)
Inventor
Sven Pedersen
Claus Christophersen
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
Novozymes AS
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 Novozymes AS, Novo Nordisk AS filed Critical Novozymes AS
Publication of EP0941359A1 publication Critical patent/EP0941359A1/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Withdrawn legal-status Critical Current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
    • 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.
  • AMGTM Novo Nordisk A/S
  • a debranching enzyme such as an isoamylase (EC 3.2.1.68) or a pullulanase (EC 3.2.1.41; e.g. PromozymeTM, from Novo Nordisk A/S) .
  • the pH of the medium is normally reduced to a value below 4.5 (e.g pH 4.3), maintaining the high temperature (above 95°C) , and the liquefying ⁇ -amylase activity is thereby denatured.
  • the temperature is then normally lowered to 60°C, and glucoamylase and debranching enzyme are ad- ded.
  • the saccharification process is normally allowed to proceed for 24-72 hours.
  • the pH of the medium is increased to a value in the range of 6-8, preferably pH 7.5, and calcium ions are removed by ion exchange.
  • the resulting syrup may then be converted into high fructose syrup using, e.g., an immobilized "glucose isomerase” (xylose isomerase, EC 5.3.1.5; e.g. SweetzymeTM, from Novo Nordisk A/S).
  • an immobilized "glucose isomerase” xylose isomerase, EC 5.3.1.5; e.g. SweetzymeTM, from Novo Nordisk A/S.
  • Enzyme classification numbers (EC numbers) referred to in the present specification with claims are in accordance with the Recommendations (1992) of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, Academic Press Inc., 1992.
  • an ⁇ -amylolytic enzyme which is stable and highly active at low concentrations of free calcium ( ⁇ 40 ppm) will be required.
  • Such an enzyme should preferably have a pH optimum at a pH in the range of 4.5-6.5, more preferably in the range of 4.5-5.5.
  • thermostable ⁇ -amylolytic enzyme which does not require a separate inactivation step.
  • One object of the present invention is to achieve improved performance of known (currently employed) ⁇ -amylolytic enzymes in relation to starch liquefaction processes by exploiting the binding properties of the CBD in question in order, for example, to modify the affinity of the starch substrate for the enzyme, and/or modify the conformation or geometry of the starch substrate, in such a manner that the course of the enzyme- catalysed reaction becomes modified in an appropriate manner.
  • 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
  • the amylolytic enzyme is D- enzyme (EC 2.4.1.25) or Q-enzyme (EC 2.4.1.18) and/or a debranching enzyme, which include isoamylase (EC 3.2.1.68) and pullulanase (or debranching enzyme) (EC 3.2.1.41).
  • a CBD and a debranching enzyme such as an isoamylase or a pullulanase
  • a debranching enzyme such as an isoamylase or a pullulanase
  • amylopectin- derived starch fragments e.g. in connection with the above- outlined saccharification stage of a starch conversion process
  • the CBD used may be in the form of e.g. a pure CBD or an enzyme comprising a CBD.
  • amylopectin is debranched with a debranching enzyme (PromozymeTM being available from Novo Nordisk) and a CBD in the form of a commercial cellulase (Carezyme being available from Novo Nordisk) , comprising a CBD is used.
  • 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 CBD used according to the method of the invention may e.g. be comprised in (i.e. part of) a cellulase, a xylanase, a mannanase, an arabinofuranosidase, an acetylesterase, a chitinase, a glucoamylase or a CGTase.
  • a further aspect of the present invention relates to the a method for recovering starch from starch-containing corn kernels by steeping the kernels in the presence of CBD and a hemicellulotic and cellulolytic activity.
  • the hemicellulolytic activity is a xylanase activity (EC 3.2.1.8, EC.3.2.1.32, EC. 3.2.1.136) and the cellulolytic activity is a cellulase activity (EC. 3.2.1.4).
  • the starch-containing corn kernels is further steeped in the presence of a pectolytic activity, such as a pectinase activity (EC. 3.2.1.15).
  • a suitable commercial product comprising these activities are SteepzymeTM from Novo Nordisk.
  • Steeping is normally performed as a pretreatment in connection with corn wet milling for the purpose of separating the corn kernels into their starch, protein (primarily gluten) , germ and fibre fractions [see, e.g., D. Ling and D.S. Jackson, Cereal Chemistry 68 (1991), pp. 205-206].
  • traditional processes which often employ steeping media containing sulfur dioxide
  • a steeping medium comprising an appropriate amount of a CBD and a xylanase leads to significantly enhanced recovery of starch from corn kernels. It also appears that the duration of the steeping procedure can be shortened in this connection. It is thus possible by this means to achieve significant savings in connection with the recovery/-isolation of starch [e.g. starch for use in liquefaction (etc.) as already outlined above].
  • the CBD is typically added in an amount of 0.01-1 gram protein per gram dry solids (DS) , preferably 0.1-0.5 gram protein per gram dry solids. Typically 1-50 FXU xylanase is added per gram dry solids (DS) .
  • the invention related to a method for separating plant materials wherein said plant material is treated with a carbohydrate-binding domain (CBD) and a xylanase.
  • CBD carbohydrate-binding domain
  • any plant material comprising xylan such as softwood and hardwood
  • CBD carbohydrate-binding domain
  • the plant material is derived from the family Poaceae (Syn: Graminaceae) and in particular prepared from a cereal such as wheat, rye, barley or oat.
  • the plant material may in addition be of vegetable or fruit origin, e.g. prepared from maize, rice, sorghum bean, or fruit hulls.
  • the plant material may be prepared from any combination of the above mentioned plants and may, in addition comprise non- plant materials.
  • the plant material to be treated according to the method of the present invention may be in any suitable form.
  • the plant material may conveniently be in the form of a pumpable dispersion or solution allowing a continuous process to be performed.
  • This dispersion is normally made by mixing dry milled material, especially wheat with a mean particle size of 50-100 mm and water.
  • the presently preferred plant material to be processed according to the invention is wheat.
  • the wheat is separated into a gluten, a starch and a fibre fraction.
  • the gluten so produced may, e.g., be added to flour in order to improve the baking properties thereof, or may be used to improve the nutritional value of products such as meat, breakfast cereals and pet food.
  • the starch may, e.g., be used for syrup production, in the paper industry, e.g. for paper coating, and in the textile industry.
  • the fibre fraction may, e.g., be used for animal feed.
  • the method of the present invention may be carried out by any industrial wheat separation process known in the art.
  • a so-called batter process or wet milling process
  • the starting material is a dilute pumpable dispersion of the wheat to be separated.
  • the dispersion is made from wheat flour and water.
  • the dry matter content of the dispersion is normally in the range of 35-50%.
  • Two major types of batter processes are known: the hydroclone process and the decanter process. These processes are advantageous in that the water consumption is relatively low.
  • the hydrocyclone process the flour is first mixed with water to make a dough, which is then further diluted and passed to an agitated agglomeration tank where gluten is agglomerated.
  • 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.
  • the decanter process differs from the hydrocyclone process in at least one major point namely when the gluten is agglomerated.
  • concentration of the batter is kept low so as to avoid that the gluten forms bigger lumps before the separation in a two-phases or three-phases decanter.
  • the mixed flour/water dispersion is pumped through a homogenised - a special pin mill with high shear forces starting the agglomeration of the gluten and just before the separation an additional dilution of the dispersion takes place.
  • the underflow contains rather clean "A"-starch and the overflow contains gluten, "B"-starch and pentosans.
  • the two phases beside the "A"-starch contains gluten with some "B"-starch and a phase with "B"-starch and pentosans.
  • the plant material separation process is normally conducted at a pH in the range of 3-8, such as 4-7 and in particular in the range of 5.5-6.5. Typically, the temperature in the range of 15-
  • the separation according to the invention is normally achieved in 1-5 minutes at a temperature of 40°C.
  • the CBD is typically added in an amount of 0.01-1 gram protein per gram dry solids (DS) , preferably 0.1-0.5 gram protein per
  • xylanase As defined herein and a cellulase
  • the cellulase may be used in an amount corresponding to 0-30,000 EGU per kg of flour, preferably in an amount corresponding to 200-5000 EGU/kg of flour.
  • a CBD in combination with a xylanase may also be used for reducing the viscosity of plant material.
  • the invention also relates to a method for reducing the viscosity of plant materials, wherein said plant material is treated with a carbohydrate-binding domain (CBD) and a xylanase.
  • CBD carbohydrate-binding domain
  • 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).
  • the invention also relates to a method for preparing wort for brewing from barley or sorghum by treating the barley or sorghum with a CBD and a xylanase. This reduces the viscosity of the wort in the brewing process.
  • the CBD and the xylanase may be used in connection with wort prepared from barley and sorghum and may be used in the same manner as pentosanases conventionally used for brewing, cf e.g. Vietor et al . , (1993) and EP 227 159.
  • Figure 1 shows the degree of debranching of amylopectin with pullulanase in the presence of CarezymeTM *
  • Figure 2 compare the degree of debranching of amylopectin with pullulanase in the presence of a purified CBD and Carezyme ⁇ , a cellulase comprising a CBD) .
  • Figure 3 shows a simplified flow sheet of the laboratory set up used in evaluating steeping using enzymes.
  • the present invention thus relates to a method for liquefying starch, wherein a starch substrate is treated in aqueous medium with a combination of an effective amount of a carbohydrate-binding domain (CBD) and at least one amylolytic enzyme, such as an ⁇ -amylase.
  • CBD carbohydrate-binding domain
  • amylolytic enzyme such as an ⁇ -amylase.
  • the amylolytic enzyme used is D-enzyme (EC 2.4.1.25) or Q-enzyme (EC.2.4.1.18) and/or a debranching enzyme, such as a pullulanase or an isoamylase.
  • a further aspect of the present invention relates to a method for saccharifying starch which has been subjected to a liquefaction process, wherein the reaction mixture after liquefaction is treated with a combination of an effective amount of a carbohydrate-binding domain (CBD) and an amylopectin- debranching enzyme (e.g. an isoamylase (EC 3.2.1.68) or a pullulanase (EC 3.2.1.41)).
  • CBD carbohydrate-binding domain
  • an amylopectin- debranching enzyme e.g. an isoamylase (EC 3.2.1.68) or a pullulanase (EC 3.2.1.41)
  • starch liquefaction processes as referred to in the context of the present invention do not embrace, for example, textile de-sizing processes wherein starch ("size") present in fabrics or textiles (normally cellulosic or cellulose-containing fabrics or textiles) is removed from the fabric or textile by an enzymatic process. It is, however, envisaged that the use of a combination of an appropriate amount of a CBD and an ⁇ -amylase will result in enhanced starch-removal performance in the context of textile de-sizing relative to that achieved using an ⁇ -amylase in the absence of the CBD.
  • Yet another aspect of the invention relates to a method for recovering starch from starch-containing corn kernels, wherein the kernels are steeped in a medium (normally predominantly aqueous) comprising a CBD and a xylanase.
  • a carbohydrate-binding domain is a polypeptide amino acid sequence which binds preferentially to a polysaccharide (carbohydrate) , frequently - but not necessarily exclusively - to a water-insoluble (including crystalline) form thereof.
  • CBDs which typically occur in chitinases
  • xylan-binding domains CBDs which typically occur in xylanases
  • mannan-binding domains CBDs which typically occur in mannanases
  • 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".
  • hydrolytic enzymes comprising a CBD - some of which have already been mentioned above - are cellulases, xylanases, mannanases, arabinofuranosidases, acetylesterases and chitinases.
  • CBDs have also been found in algae, e.g. in the red alga Porphyra purpurea in the form of a non-hydrolytic polysaccharide-binding protein [see P. Tomme et al., Cellulose- Binding Domains - Classification and Properties in Enzymatic Degradation of Insoluble Carbohydrates. John N. Saddler and Michael H. Penner (Eds.), ACS Symposium Series, No. 618 (1996) ] .
  • most of the known CBDs [which are classified and referred to by P. Tomme et al. (op cit . ) as "cellulose- binding domains"] derive from cellulases and xylanases.
  • cellulose-binding domain is intended to be understood normally in the same manner as in the latter reference (P. Tomme et al., op . cit) , and the abbreviation "CBD" as employed herein will thus often be interpretable either in the broader sense (carbohydrate-binding domain) or in the - in principle - narrower sense (cellulose- binding domain) .
  • the P. Tomme et al. reference classifies more than 120 "cellulose-binding domains" into 10 families (I-X) which may have different functions or roles in connection with the mechanism of substrate binding. However, it is anticipated that new family representatives and additional CBD families will appear in the future.
  • a CBD may be located at the N or C terminus or at an internal position.
  • 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.
  • CBDs per se as described above will typically be of relevance in the context of the invention
  • CBD-binding domain as employed in the present specification with claims may also be understood to embrace amino acid sequences, up to and including the whole of that part of the entire amino acid sequence of a CBD-containing enzyme (e.g. an enzyme such as a polysaccharide-hydrolysing enzyme) which does not include the catalytic domain of the enzyme, but which retains the CBD function of the enzyme.
  • a CBD-containing enzyme e.g. an enzyme such as a polysaccharide-hydrolysing enzyme
  • the entire amino acid sequence - comprising the catalytic function (catalytic domain) - of a enzyme may in certain respects possibly behave - at least qualitatively - in the same manner as a CBD as defined herein, such an entire amino acid sequence is not generally to be regarded as a CBD in the context of the present invention.
  • a cellulolytic enzyme cellulase
  • CBDs cellulase
  • An exception hereto will be in the case where that part of an CBD-containing enzyme • s amino acid sequence which constitutes the carbohydrate-binding domain per ⁇ e comprises the whole of the catalytic domain of the enzyme (or vice versa) or is identical thereto.
  • CBDs of interest in the context of the invention include a CBD described by Goldstein et al. [J " . Bacteriol . 175 (1993), p. 5762] and disclosed in US 5,496,934. This CBD is available from Sigma Chemical Company, St. Louis, USA, under catalogue No. C 1332.
  • Cellulases (cellulase genes ) useful for preparation of CBDs
  • cellulase refers to an enzyme which catalyses the degradation of cellulose to glucose, cellobiose, triose and/or other cello-oligosac- charides .
  • Preferred cellulases i.e. cellulases comprising preferred CBDs
  • microbial cellulases particularly bacterial or fungal cellulases.
  • Endoglucanases EC 3.2.1.4
  • monocomponent (reco binant) endoglucanases are a preferred class of cellulases, .
  • bacterial cellulases are cellulases deri- ved from or producible by bacteria from the group consisting of Pseudomonas, Bacillus, Cellulomonas, Clostridium, Microspora, Thermotoga, Caldocellum and Actinomycets such as Streptomyce ⁇ , Termomonospora and Acidothemus , in particular from the group consisting of Pseudomonas cellulolyticus, Bacillus lautus, Cellulomonas fimi, Clostridium thermocellum, in particular C.
  • the cellulase may be an acid, a neutral or an alkaline cellulase, i.e. exhibiting maximum cellulolytic activity in the acid, neutral or alkaline range, respectively.
  • a useful cellulase is an acid cellulase, preferably a fungal acid cellulase, which is derived from or producible by fungi from the group of genera consisting of Trichoderma, Myrothecium,
  • Botrytis Botrytis .
  • 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 .
  • Another useful cellulase is a neutral or alkaline cellulase, preferably a fungal neutral or alkaline cellulase, which is derived from or producible by fungi from the group of genera consisting of Aspergillus, Penicillium, Myceliophthora, Humicola, Irpex, Fusarium, Stachybotrys , Scopulariopsi ⁇ , Chaetomium, Myco- gone, Verticillium, Myrothecium, Papulo ⁇ pora, Gliocladium, Cepha- losporium and Acremonium .
  • a preferred alkaline cellulase is one derived from or producible by fungi from the group of species consisting of Humicola in ⁇ olens, Fu ⁇ arium oxy ⁇ porum, Myceliopthora thermophila, Penicillium janthinellum and Cephalo ⁇ porium sp. , preferably from the group of species consisting of Humicola in ⁇ olen ⁇ DSM 1800, Fusarium oxy ⁇ porum DSM 2672, Myceliopthora thermophila CBS 117.65, and Cephalo ⁇ porium sp. RYM-202.
  • 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) .
  • useful cellulases are variants of parent cellulases of fungal or bacterial origin, e.g. variants of a parent cellulase derivable from a strain of a species within one of the fungal genera Humicola , Trichoderma or Fu ⁇ arium.
  • proteins (protein genes) useful for preparation of CBDs are variants of parent cellulases of fungal or bacterial origin, e.g. variants of a parent cellulase derivable from a strain of a species within one of the fungal genera Humicola , Trichoderma or Fu ⁇ arium.
  • proteins (protein genes) useful for preparation of CBDs are proteins useful for preparation of CBDs
  • hydrolytic enzymes which comprise a CBD are, as already mentioned, xylanases (e.g. xylanases classified under EC 3.2.1.8 or EC 3.2.1.32), mannanases, arabinofuranosidases, acetylesterases and chitinases.
  • xylanases e.g. xylanases classified under EC 3.2.1.8 or EC 3.2.1.32
  • mannanases e.g. xylanases classified under EC 3.2.1.8 or EC 3.2.1.32
  • mannanases e.g. xylanases classified under EC 3.2.1.8 or EC 3.2.1.32
  • mannanases e.g. xylanases classified under EC 3.2.1.8 or EC 3.2.1.32
  • mannanases e.g. xylanases classified under EC 3.2.1.8 or EC 3.2.1.32
  • mannanases
  • CBDs of interest in relation to the present invention include CBDs deriving from glucoamylases (EC 3.2.1.3) or from CGTases (EC 2.4.1.19). CBDs deriving from such sources will also be generally be suitable for use in the context of one or more aspects of the invention.
  • techniques suitable for isolating e.g., xylanase genes, mannanase genes, arabinofuranosidase genes, acetylesterase genes, chitinase genes (and other relevant genes) are well known in the art.
  • 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.
  • Othet methods include the use of a selective or specific protease capable of cleaving a CBD, e.g. a terminal CBD, from the remainder of the polypeptide chain of the protein in question.
  • 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) .
  • polysaccharide-hvdrolvsing enzymes 5 Further enzymes of particular relevance for use in combination with CBDs in the context of the present invention particularly in the context of improving starch recovery from corn (maize) in corn-steeping processes (vide ⁇ upra) - include xylanases, such as those classified under EC 3.2.1.8 or EC 103.2.1.32. Chemically or genetically modified mutants of xylanases are included in this connection.
  • 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.
  • a D-enzyme may be e.g. be derived from a strain of Thermus
  • 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
  • the enzyme (s) and the CBD(s) to be used in the present invention may be in any form suited for the use in question, e.g. in the form of a dry powder or granulate, in particular a
  • non-dusting granulate a liquid, in particular a stabilised liquid, or a protected enzyme.
  • Protected enzymes may be prepared according to the method disclosed in EP 238,216.
  • Granulates may be produced, e.g., as disclosed in US 4,106,991 and US 4,661,452 (both to Novo Industri A/S), and may optionally be coated by methods known in the art.
  • the desirable levels of enzyme activity and the amount of CBD, respectively, to be used in the connection with the present invention will depend on characteristics specific to the enzyme, to the CBD and to the substrate (e.g. starch in the case of a liquefaction process) upon which the enzyme/CBD combination is to act.
  • the skilled person will be able to determine suitable dosages of enzyme activity and of CBD on the basis of methods known in the art.
  • Pullulanases Promozyme® (available from Novo Nordisk) derived from Bacillu ⁇ acidopullulyticus (described in EP 63,909).
  • Xylanase Shearzyme (available from Novo Nordisk)
  • Cellulase Carezy e® (Novo Nordisk A/S)
  • Steepzyme TM i s an experi ' mental multiactivity enzyme complex from Novo Nordisk produced from a selected strain of Aspergi •llus.
  • SteepzymeTM enzyme preparation containi•ng a number of the following activities: pectolytic, cellulolytic, and hemicellulolytic activities.
  • 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
  • the flour used in the following Examples has the following components:
  • the ⁇ -amylolytic activity of an enzyme may be determined using
  • KNU Kilo Novo ⁇ -Amylase Unit
  • 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
  • the endoxylanase activity of the sample is determined rela- tively to an enzyme standard.
  • the fermentation broths are analyzed by vibration viscosimetry on CMC at pH 6.0. More specifically, a substrate solution containing 34.0 g/1 CMC (Blanose Aqualon) in 0.1 M phosphate buffer, pH 6.0 is prepared. The enzyme sample to be analyzed is dissolved in the same buffer. 14 ml substrate solution and 0.5 ml enzyme solution are mixed and transferred to a vibration viscosimeter (e.g. MIVI 3000 available from Sofraser, France) thermostated at 40°C. Endoglucanase unit (EGU) is determined as the ratio between the viscosity of the sample and the viscosity of a standard enzyme solution.
  • a vibration viscosimeter e.g. MIVI 3000 available from Sofraser, France
  • Test conditions suitable for evaluating the performance of CBD + enzyme combinations in starch processing Test conditions (e.g. conditions of pH, temperature, calcium concentration etc.) suitable for testing, e.g., CBD + ⁇ -amylase, CBD + isoamylase or CBD + pullulanase combinations as described herein will suitably be conditions as already described above in connection with industrial starch conversion processes.
  • Assay methods suitable for determining enzymatic activity under various conditions e.g.
  • Steeping method Steeping is carried out as indicated below and in Figure 3.
  • the corn dry substance and the starch content is measured prior to steeping.
  • the germs is separated from the rest of the corn in a cylinder glass by addition of further 350 g NaCl solution and mixed. The volume after removal of the germs is about 600 ml. 54. Germ wash
  • the germ is washed with water on a vibrating screen (45 ⁇ m) until wash water stains yellow with iodine solution. The water then is recovered. The germs dry substance, amount, and starch content are measured. 105. Grinding
  • a household blender is used for blending.
  • the total blending time is 15 minutes. Blending for 1 minute is followed by 1 minute immersion in ice water. No extra water is needed. 155b. A FRYMA MZ-110 mill is used. Milling time 1 minute. Use of 1.5 1itre slurry.
  • Amylopectin (Waxy maize starch, Cerestar) was suspended in 35 deionized water to a 7 % DS slurry by stirring for 20 minutes.
  • the glass tubes were 5 well shaken and incubated at 60°C for 24 hours.
  • the molecular weight distributions were calculated using a Millenniuma 2010 Chromatography Manager, Waters with GPC option and based on pullulan standards (MW 180-1,600,000) from Polymer Labs.
  • the degree of debranching is evaluated by GPC as described and taken as the weight fraction of material with a molecular weight below 20,000 Daltons. If 40% of the material have a molecular weight below 20,000 Daltons the degree of debranching is 40%.
  • Table 2 The results in Table 2 are based on 4 repetitions. In the Figure 1 the results are showed graphically. 5 Table 2 and Figure 1 show a clear dose-response effect of the addition of Cellulase. Without Cellulase present the degree of debranching was about 40 %. With increasing amounts of cellulase the degree of debranching increased. At 0.8 mg cellulase/g DS the degree of debranching has increased to about
  • the cellulase used i.e. Carezyme , which contains a CBD, enhance the debranching efficiency of debranching enzyme.
  • An equivalent dosage of gelatine, a protein without CBD, has no or
  • Amylopectin (Waxy maize starch, Cerestar) was suspended in deionized water to a 7 % DS slurry by stirring for 20 minutes. Aliquots of 20 g of the slurry was added to glass tubes with screw caps. The starch slurries in the glass tubes were heated to 140°C for 9 minutes in an oil bath. After the gelatinisation 25 the starch solutions were air cooled to 60°C (after about 10 minutes) . After cooling the starch solutions were well shaken and then transferred to a water bath heated to 60°C.
  • CC-0017-96-D-1 0.10 mg/g DS (25 ml) 2 PUN/g DS '33 ml)
  • CC-0017-96-D-2 0.10 mg/g DS (25 ml) 2 PUN/g DS '33 ml)
  • the glass tubes were well shaken and incubated at 60°C for 24 hours. After incubation aliquots of 2 g from each glass tube were dried in small tin foil trays in an oven at 50°C over night. The resulting dry substance samples were homogenised in a mortar.
  • the molecular weight distributions were calculated using a Millenniuma 2010 Chromatography Manager, Waters with GPC option and based on pullulan standards (MW 180-1,600,000) from Polymer Labs.
  • the degree of debranching was evaluated by GPC (Gel Permeate chromotography) and taken as the weight fraction of the material with a molecular weight below 20,000 Daltons as used when evaluating the effect of Cellulase (Carezyme
  • GPC Gel Permeate chromotography
  • CBDs from Clo ⁇ tridium ⁇ teacorarium enhance the debranching efficiency of debranching enzyme.
  • the viscosity reduction provided by the combination CBD and xylanase is measured by the following method: 100 g of Fakta flour is weighed precisely. To 120 ml deionized water held at 35°C the CBD and the xylanase are added. The CBD and the xylanase are dosed as follows: Xylanase 7.5 FXU; CBD: 0.1 protein per gram Dry Solids.
  • the flour and water are stirred by hand for 30 seconds and then mixed for precisely 30 seconds on a blender (Warring, Commercial laboratory blender, Struers, Adjustments OFF 1-7, rotor in bottom
  • the viscosity at 40 rpm is measured every 15th seconds for 4 minutes.
  • the specific viscosity expressed as mean viscosity of sample/mean viscosity of blank in percents is used as a measure of the viscosity reduction.
  • the mean viscosity is a mean of the level reached after 60 seconds and until the end of measurements.
  • the wheat separation capacity of a CBD and a xylanase is evaluated by a centrifugation test: Fakta flour and water is mixed according to the procedure described in Example 3. After blending 10 ml of the batter is cen- trifugated (Megafuge 1.0 Heraeus Sepatech) at 4332 g for 5 minutes. The starch is found in the bottom layer, followed by gluten, sludge and the effluent layer at the top. The separation is expressed as an effluent percent. The higher percentage the better separation.
  • Steepzyme TM (based on corn dry substance) with and without CBD is added to the steep.

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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.
EP97912081A 1996-11-21 1997-11-21 Utilisation d'un domaine de fixation sur des glucides dans le traitement de l'amidon Withdrawn EP0941359A1 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DK133196 1996-11-21
DK133196 1996-11-21
PCT/DK1997/000537 WO1998022613A1 (fr) 1996-11-21 1997-11-21 Utilisation d'un domaine de fixation sur des glucides dans le traitement de l'amidon

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EP0941359A1 true EP0941359A1 (fr) 1999-09-15

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DE60119110T2 (de) * 2000-06-23 2006-12-14 Novozymes A/S Verfahren zum quellen
ES2517245T3 (es) 2003-06-25 2014-11-03 Novozymes A/S Enzimas para el tratamiento de almidón
WO2005045018A1 (fr) 2003-10-28 2005-05-19 Novozymes North America, Inc. Enzymes hybrides
FR2874930B1 (fr) * 2004-09-03 2008-07-25 Df3 Sarl Sarl Procede d'obtention de fractions issues du son de cereales ainsi que les fractions ainsi obtenues
CA2722889A1 (fr) 2008-04-30 2009-11-05 Danisco Us Inc. Nouveaux variants d'amylases alpha chimeriques
WO2009149395A2 (fr) 2008-06-06 2009-12-10 Danisco Us Inc., Genencor Division Alpha amylases variantes de bacillus subtilis et leurs procédés d’utilisation
JP5599113B2 (ja) 2008-06-06 2014-10-01 ダニスコ・ユーエス・インク 糖化酵素組成物及びその糖化方法
CN102112621A (zh) 2008-06-06 2011-06-29 丹尼斯科美国公司 用来自枯草芽孢杆菌的α-淀粉酶从淀粉产生葡萄糖
US9044544B2 (en) 2008-11-21 2015-06-02 Baxter International Inc. Dialysis machine having auto-connection system with roller occluder
EP3412771B1 (fr) 2009-05-19 2020-12-02 DuPont Nutrition Biosciences ApS Polypeptides d'amylase
CA2778471A1 (fr) 2009-10-23 2011-04-28 Danisco Us Inc. Procedes destines a reduire le saccharide donnant une couleur bleue

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FR2312559A1 (fr) * 1975-05-30 1976-12-24 Baxter Laboratories Inc Procede de maltage de l'orge
FI87577C (fi) * 1985-12-03 1993-01-25 Gist Brocades Nv Foerfarande foer framstaellning av voert och oel med foerbaettrad filtrerbarhet och/eller laegre viskositet
FI93859C (fi) * 1985-12-03 1995-06-12 Gist Brocades Nv Menetelmä glukoosisiirappien ja puhdistettujen tärkkelysten tuottamiseksi vehnän ja muiden viljakasvien pentosaaneja sisältävistä tärkkelyksistä
NL8702735A (nl) * 1987-11-17 1989-06-16 Dorr Oliver Inc Werkwijze voor het weken van granen met een nieuw enzympreparaat.

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See references of WO9822613A1 *

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