WO1998022613A1 - Use of carbohydrate-binding domain in starch processing - Google Patents

Abstract

The invention relates to a method for liquefying starch, wherein a starch substrate is treated in aqueous medium with a carbohydrate-binding domain (CBD) and at least one amylolytic enzyme.

Description

USE OP A CARBOHYDRATE-BINDING DOMAIN IN STARCH PROCESSING

FIELD OP THE INVENTION

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 α-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.

BACKGROUND OF THE INVENTION As indicated above, the present invention is, inter alia , of value in the field of starch processing (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.

Production of sweeteners from starch; 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. During the liquefaction process, starch (initially in the form of a starch suspension in aqueous medium) is degraded to dextrins (oligo- and polysaccharide fragments of starch) by an α-amylase [EC 3.2.1.1; e.g. Termamyl™ (Bacillus licheniformis α-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. In order to ensure optimal enzyme stability under these conditions, approximately 1 mM of calcium (ca. 40 ppm free calcium ions) is typically added to the starch suspension. After the liquefaction process the dextrins are converted into dextrose (D-glucose) by addition of a glucoamylase (amyloglucosidase, EC 3.2.1.3; e.g. AMG™, from Novo Nordisk A/S) and, typically, a debranching enzyme, such as an isoamylase (EC 3.2.1.68) or a pullulanase (EC 3.2.1.41; e.g. Promozyme™, from Novo Nordisk A/S) . Before this step 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 α-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.

After completion of the saccharification stage, 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 (dextrose 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. Sweetzyme™, 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.

From the point of view of achieving improved results in starch processing as it is "traditionally" (and currently) performed, a number of improvements in the properties of enzymes currently employed in starch conversion processes would be desirable. With respect to starch liquefaction, employing liquefying α-amylases, at least 3 improvements could be envisaged and are outlined below; each of these could be regarded as an individual benefit, although any combination (e.g. 1+2, 1+3, 2+3 or 1+2+3) could advantageously be employed: Improvement 1. Reduction of the calcium dependency of the liquefying α-amylase. Addition of free calcium (calcium ion) is required to ensure adequately high stability of α-amylases currently employed for starch liquefaction, but the presence of calcium ions in the medium at the isomerization stage results in strong inhibition of the activity of the glucoseisomerase employed therein. It is therefore necessary either to reduce the calcium ion content of the medium, by means of an expensive unit operation (e.g. ion exchange), to a level below about 3-5 ppm of free calcium, or to minimize the inhibitory effect of calcium in some other manner, e.g. by addition, after the saccharification stage, to the medium of magnesium ions in a amount sufficient to adequately "out-compete" binding of calcium to the glucoseisomerase. Significant savings could be achieved if the liquefaction process could be performed without addition of calcium ions, thereby eliminating the need for subsequent, expensive remedial unit operations to remove calcium or minimize the inhibitory effect thereof. To achieve this, an α-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.

Improvement 2. Reduction of formation of unwanted Maillard products . The extent of formation of unwanted Maillard products during the liquefaction process is dependent on the pH. Low pH favours reduced formation of Maillard products. It would thus be desirable to be able to lower the process pH from around pH 6.0 to a value around pH 4.5; unfortunately, all commonly known, thermostable liquefying α-amylases are not very stable at low pH (i.e. pH < 6.0) and their specific activity is generally low.

Achievement of the above-mentioned goal will require the availability of an α-amylolytic enzyme which is stable at a pH in the range of 4.5-5.5, and which preferably maintains a high specific activity. Improvement 3. Reduced influence of the liquefying α-amylase on the saccharification process. It has been reported previously (US patent 5,234,823) that when saccharifying with A. niger glucoamylase and B. acidopullulyticus pullulanase, the presence of residual α-amylase activity remaining after the liquefaction process can lead to lower yields of dextrose if the α-amylase is not inactivated before the saccharification stage. As already mentioned (vide supra) , this inactivation is typically carried out by adjusting the pH to below 4.5 at 95°C, before lowering the temperature to 60°C for saccharification.

The cause of this negative effect on dextrose yield is not fully understood, but it is assumed that the liquefying α-amylase preparation employed (e.g. a Termamyl™ product, such as Termamyl™ 120 L) generates "limit dextrins" (which are poor substrates for B. acidopullulyticus pullulanase) by hydrolysing 1,4-alpha-glucosidic linkages close to and on both sides of the branching points in amylopectin. Hydrolysis of these limit dextrins by glucoamylase leads to a build-up of the trisaccharide panose, which is only slowly hydrolysed by glucoamylase.

In order to avoid these problems within the framework of the process as it is currently performed, it will be necessary to develop a thermostable α-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) α-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.

SUMMARY OF THE INVENTION 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 α-amylase.

In an embodiment of the invention 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).

Similarly, and also within the scope of the invention, it is envisaged that the use of a CBD and a debranching enzyme, such as an isoamylase or a pullulanase, for debranching amylopectin- derived starch fragments (e.g. in connection with the above- outlined saccharification stage of a starch conversion process) will result in enhanced debranching performance, and thereby dextrose yield improvement, in the saccharification procedure.

In an embodiment of the invention (and as illustrated in Examples 1 and 2) the CBD used may be in the form of e.g. a pure CBD or an enzyme comprising a CBD. In Example 1 amylopectin is debranched with a debranching enzyme (Promozyme™ 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. Further, in 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

Bioengineering. 42:401-409, or by Linder M. et al. (1996),

Journal of Biological Chemistry 271:21268-21272, or in

PCT/DK97/00477 (Novo Nordisk) .

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.

Steeping 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.

In an embodiment 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).. In another embodiment 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 Steepzyme™ 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]. In traditional processes (which often employ steeping media containing sulfur dioxide) it is not possible to recover the full (analytically determined) starch content of the kernels, apparently because part of the starch remains bound in some manner to the fiber and protein components of the kernels. Without being bound by any theory, it appears that the use, in accordance with the invention, of 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) .

Method for separating plant material In a further aspect 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. While any plant material comprising xylan (such as softwood and hardwood) may be treated with CBD and a xylanase according to the method of the invention it is preferred that 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. As it will be further explained below, 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. By the process of the invention 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.

In the following the method of the invention for separation of a plant material will be described with reference to wheat separation. However, it will be understood that separation of any of the other types of plant material mentioned above may be performed by a similar type of process and the person skilled in the art would know which type of process to select for separation of a given plant material, cf, for instance, the book entitled "Starch production technology", Ed. by J.A. Radley. In fig. 2 a flow sheet illustrating a wheat separation process is shown.

The method of the present invention may be carried out by any industrial wheat separation process known in the art. However, it is presently preferred to use a so-called batter process (or wet milling process) , in which the starting material is a dilute pumpable dispersion of the wheat to be separated. Normally, 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. In 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. In the decanter process it is very important that the 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. Before the separation 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. In case of a two-phases decanter the underflow contains rather clean "A"-starch and the overflow contains gluten, "B"-starch and pentosans. For three-phases decanters the two phases beside the "A"-starch contains gluten with some "B"-starch and a phase with "B"-starch and pentosans.

When a CBD and a xylanase is used for the separation of wheat it is possible to obtain an improved capacity of dough mixing and homogenization, an improved separation capacity, a reduced viscosity in the pentosan fraction (which reduces energy consumption when evaporating and drying) and a reduced ampere consumption on decanters. Furthermore, end products of a higher 5 purity may be obtained and the processing time may be reduced due to the increased flow enabled by the reduced viscosity.

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-

1050°C such as 35-45°C. In the wheat separation process, 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

15 gram dry solids.

Typically 1-50 FXU xylanase is added per gram dry solids (DS) .

For some purposes it may be advantageous to use another enzyme together with the xylanase. For instance, it has been found that the combined use of xylanase as defined herein and a cellulase

20 has a synergistic effect. 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.

Viscosity reduction of plant material

25 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.

30 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).

35

Brewing

Further, 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.

BRIEF DESCRIPTION OP THE DRAWING

Figure 1 shows the degree of debranching of amylopectin with pullulanase in the presence of Carezyme™^*

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.

DETAILED DISCLOSURE OP THE INVENTION In a first aspect 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 α-amylase. In an embodiment of the invention 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)). It is to understood that 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 α-amylase will result in enhanced starch-removal performance in the context of textile de-sizing relative to that achieved using an α-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.

Carbohydrate-binding domains

A carbohydrate-binding domain (CBD) 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.

Although a number of types of CBDs have been described in the patent and scientific literature, the majority thereof - many of which derive from cellulolytic enzymes (cellulases) - are commonly referred to as "cellulose-binding domains"; a typical cellulose-binding domain will thus be a CBD which occurs in a cellulase. Likewise, other sub-classes of CBDs would embrace, e.g., chitin-binding domains (CBDs which typically occur in chitinases) , xylan-binding domains (CBDs which typically occur in xylanases) , mannan-binding domains (CBDs which typically occur in mannanases) , and others.

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. 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". Examples of 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) ] . However, 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.

In the present context, the term "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.

In proteins/polypeptides in which CBDs occur (e.g. enzymes, typically hydrolytic enzymes) , 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. For example: 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, whilst one CBD (derived from a cellulase from Clostridium thermocellum) listed and classified in Family VII consists of 240 amino acid residues. Accordingly, 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.

Although CBDs per se as described above will typically be of relevance in the context of the invention, the term "carbohydrate-binding domain" (CBD) 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.

Thus, while the entire amino acid sequence - comprising the catalytic function (catalytic domain) - of a enzyme [e.g. a cellulolytic enzyme (cellulase) , or another enzyme comprising one or more CBDs] 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. 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 εe comprises the whole of the catalytic domain of the enzyme (or vice versa) or is identical thereto.

Commercially available 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

Techniques suitable for isolating a cellulase gene are well known in the art. In the present context, the term "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) in the present context are microbial cellulases, particularly bacterial or fungal cellulases. Endoglucanases (EC 3.2.1.4), particularly monocomponent (reco binant) endoglucanases, are a preferred class of cellulases, .

Useful examples of 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 Streptomyceε , Termomonospora and Acidothemus , in particular from the group consisting of Pseudomonas cellulolyticus, Bacillus lautus, Cellulomonas fimi, Clostridium thermocellum, in particular C. stercorarium, Microspora bispora, Termomonospora fusca, Termomonospora cellulolyticum and Acidothemus cellulolyticus . 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,

Aspergillus, Phanaerochaete , Neurospora, Neocallimastix and

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 , Scopulariopsiε, Chaetomium, Myco- gone, Verticillium, Myrothecium, Papuloεpora, 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 inεolens, Fuεarium oxyεporum, Myceliopthora thermophila, Penicillium janthinellum and Cephaloεporium sp. , preferably from the group of species consisting of Humicola inεolenε DSM 1800, Fusarium oxyεporum DSM 2672, Myceliopthora thermophila CBS 117.65, and Cephaloεporium 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 inεolenε DSM 1800, or which is a derivative of the latter ~43kD endoglucanase and exhibits cellulase activity (e.g. Carezyme™) .

Other examples of 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 Fuεarium. Other proteins (protein genes) useful for preparation of CBDs

Examples of other types of 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. As also mentioned previously, CBDs have also been found, for example, in certain algae, e.g. in the red alga Porphyra purpurea in the form of a non-hydrolytic polysaccharide-binding protein. Reference may be made to P. Tomme et al. (op cit . ) for further details concerning sources (organism genera and species) of such CBDs. Further 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. In this connection, 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.

Isolation of a CBD

In order to isolate 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. These gene- deletion methods result in a mutated gene encoding a shortened gene molecule whose expression product may then be evaluated for substrate-binding (e.g. cellulose-binding) ability. Appropriate substrates for evaluating the binding ability include cellulosic materials such as Avicel™ 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 The term "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 α-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 α-amylases include, for example, α- amylases obtainable from Bacilluε species, in particular a special strain of B. licheniformis, described in more detail in GB 1296839. Relevant commercially available amylases include

Duramyl™, Termamyl™, Fungamyl™ and BAN™ (all available from Novo Nordisk A/S, Bagsvaerd, Denmark) , and Rapidase™ and Maxamyl P™ (available from Gist-Brocades, Holland) .

Starch- or starch-fragment-debranching enzymes

Isoamylases: 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 Pεeudomonaε species, (e.g- Pεeudomonaε sp. SMP1 or P. amyloderomoεa SB15) , Bacilluε species (e.g. B. amyloliquefacienε) , Flavobacterium species or Cytophaga (Lysojbaσter) species.

Pullulanases: pullulanases (EC 3.2.1.41) 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 Bacilluε species (e.g. B. acidopullulyticuε ; such as Promozyme™, from Novo Nordisk A/S) .

Other 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 εupra) - 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. An example of a relevant xylanase i •s ShearzymeTM avai•lable from Novo Nordisk A/S, or Spezyme® CP available from Genencor, USA, a H. inεolenε xylanase (produced as described in Example 2 of WO 92/17573 ,

15 Xylanase I powder (produced using Xylanase I (described in WO 94/21785) as the starting material, by solid liquid separation, concentration and freeze drying following standard methods) , Xylanase II (produced as described in WO 94/21785) , a xylanase produced by B. pumiluε strain DSM 6124 as described in WO

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

25 thermophiluε , Thermococcuε lithoraliε , Cloεtridium butyricum , Streptococcuε pneumoniae , E . coli or from Solanum tuberosum (potato) .

If appropriate, more than one CBD (e.g. selected among those types of CBDs mentioned herein) may be used in

30 combination with more than one enzyme (e.g. two or more enzymes selected among the types of enzymes mentioned herein) .

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

35 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.

Materials and Methods

Pullulanases: Promozyme® (available from Novo Nordisk) derived from Bacilluε 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 εtercorarium (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 εtercorarium 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 Cloεtridium εtercorarium) . Amylopectin (Waxy maize starch, Cerestar) Flour

The flour used in the following Examples has the following components:

5 Fakta flour: a commercial flour of non-specified type ("Luksus hvedemel", prepared by Dagligvaregruppen, DK-7100 Vejle) .

Determination of α-amylolvtic activity (KNU)

The α-amylolytic activity of an enzyme may be determined using

10 potato starch as substrate. This method is based on the breakdown (hydrolysis) of modified potato starch, and the reaction is followed by mixing samples of the starch/enzyme or starch/hybrid enzyme solution with an iodine solution. Initially, a blackish- blue colour is formed, but during the break-down of the starch

15 the blue colour becomes weaker and gradually turns to a reddish- brown. The resulting colour is compared with coloured glass calibration standards.

One Kilo Novo α-Amylase Unit (KNU) is defined as the amount of enzyme (activity) which, under standard conditions (i.e. at

2037±0.05°C, 0.0003 M Ca²⁺, pH 5.6) dextrinizes 5.26 g starch dry substance (Merck Amylum solubile) . Determination of pullulanase activity fPUN)

Activity determination One Pullulanase Unit Novo (PUN) is defined as the amount of enzyme which hydrolyzes pullulan, liberating reducing carbohydrate with a reducing power equivalent to 1 micro-mol glucose per minute under the following standard conditions:

Substrate: 0.2% pullulan

Temperature: 40°C pH: 5.0

Reaction time: 30 minutes

A detailed description of the analysis method (AF 190) is available on request.

Determination of xylanase activity (FXU1

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.

The endoxylanase activity of the sample is determined rela- tively to an enzyme standard.

A detailed description of Novo Nordisk' s method of analysis is available on request.

Determination of endo-glucanase activity (EGU)

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.

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 + α-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. pH, temperature, calcium concentration etc., depending on the nature of the enzyme hybrid) are well known in the art for numerous types of enzymes which are appropriate for use in combination with a CBD as described herein, and a person of ordinary skill in the art will readily be able to select assay procedures suitable for evaluating the enzymatic performance of such combinations as employed in the present context.

Steeping method Steeping is carried out as indicated below and in Figure 3.

1. Steeping.

The corn dry substance and the starch content is measured prior to steeping.

During steeping the pH, dry substance, and amount of dry substance is determined.

2. Degermination.

The steeped corn kernals are blended gently. 3. Flotation

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 μm) 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

5a. 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.

6. Fibre wash

Wash with water on vibrating screen (45μm) until wash water stains yellow with iodine solution. Measurement of fibres 20 dry substance, amount, and starch content.

7. Starch/Gluten isolation.

Collect slurry throughput from fibre wash on Biichner funnel placed on top of a vacuum flask where vacuum is applied. Measurement of starch/gluten dry substance, amount, and starch 25 content. Wash water measurement of amount and dry substance. Mass balance on material dry substance basis. Mass balance on starch.

EXAMPLES

30

EXAMPLE 1

Debranching of amylopectin with a debranching enzyme and

Carezymef

Amylopectin (Waxy maize starch, Cerestar) was suspended in 35 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 gelatinization 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. 5 Except for 2 glass tubes (control tubes) , a basic dosage of 2

PUN/g DS of debranching enzyme (Promozyme TM 600 L, batch AGN

2008; correspond to 5 ml/glass tube) were added to all tubes. Gelatine and cellulase (Carezyme™* were added to the starch solutions according to the scheme below: 0

*) Purified Cellulase, batch car-wt-9509, 9.8 mg enzyme protein/ml (e.p.= enzyme protein) π) Gelatine, Merck 4078, diluted to 10 mg/ml

After addition of enzymes and gelatine the glass tubes were 5 well shaken and incubated at 60°C for 24 hours.

After the incubation the content in the glass tubes 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. 0 20 mg of each dried sample were dissolved in 8 ml DMSO

(dimethylsulfoxide) by heating to 70°C overnight in a water bath. The solutions were filtered through a 1 mm filter (Gelman

Acrodi •scTM CR PFTE) . The GPC analyses were carri•ed out on Waters equipment (pump, autosampler and RI detector) using 3 5 PLGel 20 mm MIXED A 300 x 7.5 mm in series from Polymer Labs., UK. The samples were eluted with 99.9 % (v/v) DMSO and 0.1 % 0.01 M NaNθ3 using a flow rate of 0.5 ml/min. The columns were heated to 40°C. 100 ml samples were analysed.

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 experiments showed that a dosage of about 2 PUN/g DS of debranching enzyme give a degree of debranching of about 40-

50 %. This dosage of debranching enzyme was chosen so that it gave a reasonable degree of debranching and still allowed for further enhancement.

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%.

Different dosages of gelatine (0.25, 0.5 and 0.75 mg/g DS as described) were incubated with debranching enzyme (2 PUN/g DS) in order to see a possible effect of protein. In table 1 the degree of debranching is listed for the different dosages of gelatine. Table 1

basis dosage of debranching enzyme of 2 PUN/g DS.

From the results in Table 1 there appears to be no effect of addition of gelatine during debranching of amylopectin with debranching enzyme. Up to a gelatine dosage of 0.5 mg/g DS the degree of debranching is about 52 % and at 0.75 mg/g DS it is about 47 %. If gelatine has any effect on the efficiency of debranching it appears to be negative at high dosages. Table 2 show the results from debranching with 2 PUN/g DS of debranching enzyme with different dosages of cellulase (comprising a CBD) . 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

1055 %, corresponding to an enhancement of the debranching efficiency of 38 %.

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

15 a little negative effect on the debranching efficiency.

Example 2

Debranching of amylopectin with a debranching enzyme and a pure CBD 20 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. debranching enzyme (Promozyme 600 L, batch AGN 2008; diluted to 60 PUN/ml) and CBDs (Cloεtridium εteacorarium , batch no. opr 30051195, MB 206#9644, 4 mg/ml) were added to the starch solutions according to the scheme below:

Sample ID Dosage of CBD (amount) Dosage of debranching enzyme 'amount )

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)

CC-0017-96-D-3 0.30 mg/g DS (75 ml) 2 PUN/g DS '33 ml)

CC-0017-96-D-4 0.30 mg/g DS (75 ml) 2 PUN/g DS [33 ml)

CC-0017-96-D-5 0.50 mg/g DS (125 ml) 2 PUN/g DS 33 ml)

CC-0017-96-D-6 0.50 mg/g DS (125 ml) 2 PUN/g DS '33 ml)

CC-0017-96-D-7 0.70 mg/g DS (125 ml) 2 PUN/g DS < '33 ml)

CC-0017-96-D-8 0.70 mg/g DS (125 ml) 2 PUN/g DS '33 ml)

CC-0017-96-D-9 0.0 mg/g DS (0 ml) 2 PUN/g DS '33 ml)

CC-0017-96-D-10 0.0 mg/g DS (0 ml) 2 PUN/g DS '33 ml)

CC-0017-96-D-11 0.0 mg/g DS (0 ml) 3 PUN/g DS '50 ml)

CC-0017-96-D-12 0.0 mg/g DS [0 ml) 3 PUN/g DS 50 ml)

CC-0017-96-D-13 0.0 mg/g DS [0 ml) 4 PUN/g DS | '67 ml)

CC-0017-96-D-14 0.0 mg/g DS [0 ml) 4 PUN/g DS ( '67 ml)

CC-0017-96-D-15 0.0 mg/g DS (0 ml) 5 PUN/g DS ( 83 ml)

CC-0017-96-D-16 0.0 mg/g DS [0 ml) 5 PUN/g DS ( 83 ml)

CC-0017-96-D-17 0.0 mg/g DS (0 ml) 0 PUN/g DS ( 0 ml)

After addition of the debranching enzyme and CBD 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.

20 mg of each dried sample were dissolved in 8 ml dimethylsulfoxide (DMSO) by heating to 70°C overnight in a water bath. The solutions were filtered through a 1 mm filter (Gelman Acrodisc™ CR PFTE) . The GPC analyses were carried out on Waters equipment (pump, autosampler and RI detector) using 3 PLGel 20 mm MIXED A 300 x 7.5 mm in series from Polymer Labs., UK. The samples were eluted with 99.9 % (v/v) DMSO and 0.1 % 0.01 M NaNθ3 using a flow rate of 0.5 ml/minutes. The columns were heated to 40°C. 100 ml samples were analysed. 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|) (Example 1) . In Table 3 the degree of debranching for different dosages of debranching enzyme is listed.

Table 3

From Table 3 it is seen that going from 2 PUN/g DS to 5 PUN/g DS increases the degree of debranching from 37 % to 44 %.

In Table 4 the degree of debranching for different dosages of CBD using a constant dosage of 2 PUN/g DS of debranching enzyme is listed. Table 4

From Table 4 it is seen that an increasing amount of CBD increases the efficiency of debranching enzyme slightly.

Though there are some variances among the results at 0.5 and 0.7 mg/g DS the efficiency of the pure CBDs are not so pronounced as for Carezyme| (comprising a CBD) dosed at the same mg level. This is illustrated in Figure 2, where the dose- response curves for the pure CBDs and for the cellulase comprising a CBD (i.e. Carezyme ) (results from Example 1) are shown.

The value of 53 % at 0.5 mg/g DS has not been used in the figure. As can be seen CBDs from Cloεtridium εteacorarium enhance the debranching efficiency of debranching enzyme.

EXAMPLE 3

Viscosity reduction by CBD and Xylanase

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.

A sample comprising 7.5 FXU xylanase only and a blank sample is used as control (no CBD and no xylanase added) are also tested.

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

(4 blades)) at 7 (maximum speed). It lasts 30 seconds to pour the liquid into the measuring tube at the viscometer (Programmable rheometer, model DV-111, Brookfield, Spindel 25, the measuring tube being thermostated at 38°C) . 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.

EXAMPLE 4 Wheat separation

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.

EXAMPLE 5 Steeping of corn kernels

A method for steeping of starch-containing corn kernels is described above in the "Steeping method" section and further in Figure 3.

0.2% Steepzyme TM (based on corn dry substance) with and without CBD is added to the steep.

Simultaneous addition of Steepzyme™ and CBD give a higher starch yields, less starch in germs and fibres, lower viscosity and less sludge in the steepwater, less fibres in downstream operation, reduced steeping time compared to a steeping trials wi •th addi•ti•on of SteepzymeTM alone.

Claims

1. A method for liquefying starch, wherein a starch substrate is treated in aqueous medium with a carbohydrate-binding domain

5 (CBD) and at least one amylolytic enzyme.

2. The method according to claim 1 , wherein a starch substrate is treated in aqueous medium with a carbohydrate-binding domain (CBD) and an α-amylase.

10

3. The method according to claim 1, wherein a starch substrate is treated in aqueous medium with a combination of a carbohydrate- binding domain (CBD) and D-enzyme or Q-enzyme and/or a debranching enzyme.

15

4. The method according to claim 3 , wherein the debranching enzyme is a pullulanase or an isoamylase.

5. A method for saccharifying starch which has been subjected to 20 a liquefaction process, wherein the reaction mixture after liquefaction is treated with a carbohydrate-binding domain (CBD) and an amylopectin-debranching enzyme

6. The method according to claim 5, wherein said amylopectin 25 debranching enzyme is an isoamylase or a pullulanase.

7. The method according to claim 5 , wherein the CBD used is in the form of an enzyme comprising one or more CBDs.

308. The method according to claim 7, wherein the enzyme comprising a CBD is a cellulase, a xylanase, a mannanase, an arabinofuranosidase, an acetylesterase, a chitinase, a glucoamylase or a CGTase.

359. 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 a carbohydrate- binding domain (CBD) and a glucoamylase.

10. The method according to any of claims 5-9, wherein the starch substrate has been liquefied by treatment with an α-amylase.

5

11. The method according to any of claims 5-9, wherein the liquefaction of the starch substrate has been performed according to claim 1-4.

1012. A method for recovering starch from starch-containing corn kernels, wherein said kernels are steeped in aqueous medium comprising a CBD and a hemicellulolytic and a cellulolytic activity.

1513. The method according to claim 12, wherein the hemicellulolytic activity is a xylanase activity.

14. The method according to claim 12 , wherein the cellulolytic activity is a cellulase activity.

20

15. The method according to claim 12-14, wherein the starch- containing corn kernels further is steeped in the presence of a pectolytic activity.

2516. The method according to claim 15, wherein the pectolytic activity is a pectinase activity.

17. A method for reducing the viscosity of plant materials, wherein said plant material is treated with a carbohydrate-

30 binding domain (CBD) and a xylanase.

18. A method for separation plant materials, wherein said plant material is treated with a carbohydrate-binding domain (CBD) and a xylanase.

35

19. The method according to any of claims 17-18, wherein the plant material is wheat, rye, barley or oat.

20. A method for preparing wort for brewing from barley or sorghum wherein the barley or sorghum is treated with a CBD and a xylanase.

21. The method according to any one of the preceding claims, wherein said CBD is a CBD deriving from a cellulase, a xylanase, a mannanase, an arabinofuranosidase, an acetylesterase, a chitinase, a glucoamylase or a CGTase.