1. A proceoss for producing dextrose from starch by saooharification comprising the steps of liquifying starch by known means and adding a pure carbohydrate binding donain (CBD) and atleast one anylopeotin-debranching enyme, to said liquified starch and recovering dextrose from the reaction mixture by method known per se.

2. The process as claimed in claim 1, wherein said auylopeotin-debranching enzyme is an isoanylase or a pullulanase.

3. The process as claimed in claim 1, wherein said amylopectln debranohlng enzyne is a gluooanylase.

4. The process as claimed in clains 1-3, wherein starch is liquified by treatment with an -amylase.

5. The process as claimed in claims 1-3, wherein starch is liquified by treatment with a combination of pure carbohydrate binding domain and atleast one amylolytlc enzyme between pH 5.5 and 8.2 at a temperature range of 95°C to 180°C.

6. A process for producing dextrose from staroh by saooharification substantially as herein described.

This invention relates to a process for producing dextrose from starch by saccharifioation.
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 sacoharlfioation processes are described in, e.g., US 3,912, 580 and in EP 0 252 730 and EP 0 063 909.
Production of sweeteners fron starch: A "traditional" process for the production of glucose- and fructose-containing syrupa from starch nornally consists of three conseoutive enzymatic processes, viz. a liquefaction process followed by a saccharifioation process and (for production of fruotose-containing syrups) an isomerization process. During the liquefactions process, starch (initially in the form of a starch suspension in aqueous medium) is degraded to dextrin’s (oligo- and polysaccharide fragments of starch) by an (-amylase [EC 3.2.1.1; e.g. TermanylTM(Bacillus lichenifornis -amylase), available from Novo Nordisk A/S, Bagsvaerd, Denmark], typically at pH values between 5.5 and 8.2 and at temperatures of 95-180° 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 eure 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 disbranching enzyme, such as an isoamylase (EC 3.2.1.68) or a pullulanase (EC 3.2.1.41; e.g. Proraozyme™, 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 a-amylase activity is thereby denatured. The temperature is then normally lowered to 60°C, and glucoamylase and disbranching 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’s" (xylene isomerase, EC 5.3.1.5; e.g. Sweetly™, from Novo Nor disk A/S).
Enzyme classification numbers (EC numbers) referred to in the present specification with claims are in accordance with the Recommendations (l992) 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 a-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 g-amylase. Addition of free calcium (calcium ion) is required to ensure adequately high stability of a-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 3ome 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 cal-:inure ions, thereby eliminating the need for subsequent, expensive remedial unit operations to remove calcium or minimize the inhibitory effect thereof.
To achieve this, an a-amylolytic enzyme which is stable and lightly active at low concentrations ~bf ~ free calcium (< 40 ppm) will be required. Such an enzyme should preferably have a pH jptimum 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 luring the liquefaction process is dependent on the pH. Low pH 'amours 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, thermos table liquefying a-cunylases 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 a-amylolytic enzyme which is stable at a pH in the range of 4.5-5.5, and which preferably maintains a high spe-trafic activity.

Improvement 3. Reduced Influence of the liquefying g-amvlase on the saccharification process. It has been reported previously (US patent 5,234,823) that when saccharifying with A. niger. glucoamylase and B. acidopullulytxcus pullulanase, the presence of residual a-amylase activity remaining after the liquefaction process can lead to lower yields of dextrose if the a-amylase is not inactivated before the saccharification stage. As already mentioned (vide supra), this inactivation is typically carried put by adjusting the pH to below 4.5 at 95"C, before lowering the temperature to SO^C for saccharification.
The cause of this negative effect on dextrose yield is not fully understood, but it is assumed that the liquefying a-amylase prepare action employed (e.g. a Termamy l^'* product, such as Perm amyl™ 120 L) generates "limit dextrin" (which are poor substrates for B. acidopallulyticus pullulanase) by hydrolyzing .,4-alpha-glucosidic linkages close to and on both sides of the iranching points in amyl pectin. Hydrolysis of these limit lextrins by glucoamylase leads to a build-up of the disaccharides lanose, which is only slowly hydrolysed by glucoeunylase.
In order to avoid these problems within the framework of the process as it is currently performed, it will be necessary to evelop a thermos table a-amylolytic enzyme which does not require separate inactivation step.
One object of the present invention is to achieve improved performance of known (currently employed) a-amylolytic enzymes in elation to starch liquefaction processes by exploiting the ending properties of the CBD in question in order, for example, modify the affinity of the starch substrate for the enzyme, and/or modify the conformation or geometry of the starch abstracter, in such a manner that the course of the enzyme-analyzed reaction becomes modified in an appropriate manner.
SUMMARY OF THE INVENTION
One aspect of the invention relates to an improved enzymatic rocess for liquefying starch employing a combination of a orbohydrate-binding domain (CBD; vide infra) and at least one

liquefying amylolytic enzyme, such as an a-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 Rebranching 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 in isoamylase or a pullulanase, for debranching amylopectin-lerived starch fragments (e.g. in connection with the above-lutlined saccharification stage of a starch conversion process) dll result in enhanced debranching performance, and thereby .extrose yield improvement, in the saccharification procedure.
In an embodiment of the invention (and as illustrated in xamples 1 and 2) the CBD used may be in the form of e.g. a ure CBD or an enzyme comprising a CBD. In Example 1 mylopectin is debranched with a debranching enzyme Promozyme^" being available from Novo Nordisk) and a CBD in he form of a commercial cellulase (Carezyme being available rom Novo Nordisk), comprising a CBD is used. Further, in sample 2 a pure CBD di-mer derived from Clostridium tercorarium (NCIMB 11754) XynA (GenBank and SWISS-PROT ::cession No. 13325) is used in combination with a debranching izyme (Promozyme'^") for debranching amylopectin. The pure CBD ly be provided using techniques well-known in the art, e.g. as iscribed by Ong E. et al. (1993), Biotechnology and .oengineering. 42:4 01-409, or by Linder M. et al. (1996) , surnal of Biological Chemistry 271:21268-21272, or in :T/DK97/00477 (NOVO Nordisk).
le CBD used according to the method of the invention may e.g. be tmprised in (i.e. part of) a cellulase, a xylanase, a mannanase, arabinofuranosidase, an acetylesterase, a chitinase, a ucoamylase or a CGTase.
eepinq
A further aspect of the present invention relates to the a thod for recovering starch from starch-containing corn rnels by steeping the kernels in the presence of CBD and a

hemicellulotic and cellulolytic activity.
Cn an embodiment the hemicellulolytic activity is a xylemase ictivity (EC 3.2.1.8, EC.3.2.1.32, EC. 3.2.1.136) and the ;ellulolytic activity is a cellulase activity (EC. 3.2.1.4).. . :n another embodiment the starch-containing corn kernels is ■virther steeped in the presence of a pectolytic activity, such as I pectinase activity (EC. 3.2.1.15). A suitable commercial iroduct comprising these activities are Steepzyme^'* from Novo tordisk.
Steeping is normally performed as a pretreatment in :onnection with corn wet milling for the purpose of separating :he corn kernels into their starch, protein (primarily gluten), rerm and fibre fractions [see, e.g., D. Ling and D.S. Jackson, •ereal Chemistry 68 (1991), pp. 205-206]. In traditional rocesses (which often employ steeping media containing sulfur ioxide) it is not possible to recover the full (analytically etermined) starch content of the kernels, apparently because art of the starch remains bound in some manner to the fiber nd protein components of the kernels.
Without being bound by any theory, it appears that the use, n accordance with the invention, of a steeping medium omprising an appropriate amount of a CBD and a xylanase leads o significantly enhanced recovery of starch from corn kernels, t also appears that the duration of the steeping procedure can e shortened in this connection. It is thus possible by this eans to achieve significant savings in connection with the ecovery/-isolation of starch [e.g. starch for use in iquefaction (etc.) as already outlined above], he CBD is typically added in an amount of o.oi-l gram protein er gram dry solids (DS), preferably 0.1-0.5 gram protein per cam dry solids. Typically 1-50 FXU xylanase is added per gram ry solids (DS).
athod for separating Plant material
In a fxirther aspect the invention related to a method for eparating plant materials wherein said plant material is ceated with a carbohydrate-binding domain (CBD) and a xylanase.

While ciny plant material comprising xylan (such as softwood and ardwood) may be treated with CBD and a xylanase according to the lethod of the invention it is preferred that the plant material s derived from the family Poaceae (Syn: Grarainaceae) and in. articular prepared from a cereal such as wheat, rye, barley or at. The plant material may in addition be of vegetable or fruit rigin, e.g. prepared from maize, rice, sorghiun bean, or fruit alls. The plant material may be prepared from any combination of he above mentioned plants and may, in addition comprise non-lant materials.
The plant material to be treated according to the method of the resent invention may be in any suitable form. As it will be urther explained below, the plant material may conveniently be n the form of a ptunpable dispersion or solution allowing a ontinuous process to be performed. This dispersion is normally ade by mixing dry milled material, especially wheat with a mean article size of 50-100 nun and water.
The presently preferred plant material to be processed ccording to the invention is wheat. By the process of the nvention the wheat is separated into a gluten, a starch and a ibre fraction. The gluten so produced may, e.g., be added to Lour in order to improve the baking properties thereof, or may = used to improve the nutritional value of products such as 3at, breakfast cereals cmd pet food. The starch may, e.g., be sed for syrup production, in the paper industry, e.g. for paper Dating, and in the textile industry. The fibre fraction may, .g., be used for animal feed. In the following the method of the invention for separation of
plant material will be described with reference to wheat aparation. However, it will be understood that separation of any E the other types of plant material mentioned above may be srformed by a similar type of process and the person skilled in le art would know which type of process to select for separation f a given plant material, cf, for instance, the book entitled Starch production technology", Ed. by J.A. Radley. In fig. 2 a Low 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 nilling process), in which the starting material is a dilute pumpable dispersion of the wheat to be separated. Normally, the. iispersion is made from wheat flour and water. The dry matter content of the dispersion is normally in the range of 35-50%. Two lajor types of batter processes are known: the hydroclone process ind the decanter process. These processes are advantageous in Jiat the water consumption is relatively low.
In the hydrocyclone process the flour is first mixed with water
:o make a dough, which is then further diluted and passed to an
gitated agglomeration tank where gluten is agglomerated. The
ispersion 'with the small gluten agglomerates and starch is
imped to a set of hydrocyclones, where a centrifugal separation
akes place. The gluten and the "B"-starch being the lightest
raction leaves the top of the hydrocyclones together and the
luten is separated from the "B"-starch by screens. The underflow
rem the hydrocyclones consists mainly of "A"-starch, while
Bntosan (or fibres) are found in both fractions. The fractions
re further cleaned by a series of washing/concentration steps.
The decanter process differs from the hydrocyclone process in
: least one major point neimely when the gluten is agglomerated.
1 the decanter process it is very important that the concen-
:ation of the batter is kept low so as to avoid that the gluten
irms bigger lumps before the separation in a two-phases or
iree-phases decanter. Before the separation the mixed
.our/water dispersion is pumped through a homogenised - a
lecial pin mill with high shear forces starting the
[glomeration of the gluten and just before the separation an
Iditional dilution of the dispersion takes place. In case of a
o-phases decanter the underflow contains rather clean
."-starch and the overflow contains gluten, "B"-starch and
ntosans. For three-phases decanters the two phases beside the
"-starch contains gluten with some "B"-starch and a phase with
"-starch and pentosans.
When a CBD and a xylanase is used for the separation of wheat
is possible to obtain an improved capacity of dough mixing and

tiomogenization, an improved separation capacity, a reduced iriscosity in the pentosan fraction (which reduces energy consumption when evaporating and drying) eind a reduced cunpere consumption on decanters. Furthermore, end products of a higher purity may be obtained and the processing time may be reduced due lo the increased flow enabled by the reduced viscosity.
The plant material separation process is normally conducted at i 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 15-50 "C such as 35-45 "C. In the wheat separation process, the separation according to the invention is normally achieved in 1-5 ninutes at a temperature of 40''C.
[■he CBD is typically added in an amount of o.oi-l gram protein 3er gram dry solids (DS), preferably 0.1-0.5 gram protein per jram 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 Jie combined use of xylanase as defined herein and a cellulase las a synergistic effect. The cellulase may be used in an amount jorresponding to 0-30,000 EGU per kg of flour, preferably in an imount corresponding to 200-5000 EGU/kg of flour.
viscosity reduction of plant material
A CBD in combination with a xylanase may also be used for •educing the viscosity of plant material.
The invention also relates to a method for reducing the riscosity of plant materials, wherein said plant material is created with a carbohydrate-binding domain (CBD) and a xylanase.
The viscosity reduction may be important, e.g. in a continuous rheat separation process, in that an increased wheat flour flow lay be obtained. Furthermore, the viscosity reduction is .mportant in the preparation of food or feed and in brewing, cf '^isser et al., Xylans and Xylanases, (1991).
trewing
Further, the invention also relates to a method for preparing

wort for brewing from barley or 8orghu» by treating the barley or sorghum with a CBD end 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 oanner as pentoaanases conventionally used for brewing, cf e.g. Vietor et al., (1993) and EP 227 156.
Accordingly the present invention provides a process for producing dextrose from starch by oaccharification comprising the steps of liquifying starch by known means and adding a pure carbohydrate binding domain (CBD) and atleast one amylopectin-debranchlng enzyme, to said liquified starch and recovering dextrose fron the reaction mixture by method known per se.
With reference to the aooompanylng drawings:
Figure 1 shows the degree of debranohing of amylopectin with pullulanase in the presence of Carezyme.
Figure 2 compares the degree of debranohing 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 et up used in evaluating steeping using enzymes.
ETAILED DISCLOSnSK OF THE INVENTION
In a first aspect the present invention thus relates to a ethod for liquefying starch, wherein a starch substrate is reated in aqueous mediiun with a combination of an effective mount of a carbohydrate-binding domain (CBD) and at least one nylolytic enzyme, such as an a-amylase.
In an embodiment of the invention the amylolytic enzyme used 3 D-enzyme (EC 2.4.1.25) or Q-enzyme (EC.2.4.l.18) and/or a ^branching enzyme, such as a pullulanase or an isoamylase.
A further aspect of the present invention relates to a method 3r saccharifying starch which has been subjected to a Lquefaction process, wherein the reaction mixture after Lquefaction is treated with a combination of an effective amount r a carbohydrate-binding domain (CBD) and an amylopectin-ibranching enzyme (e.g. an isoamylase (EC 3.2.1.68) or a illulanase (EC 3.2.1.41)).
It is to understood that starch liquefaction processes as if erred to in the context of the present invention do not ibrace, for example, textile de-sizing processes wherein starch size") present in fabrics or textiles (normally cellulosic or illulose-containing fabrics or textiles) is removed from the brie or textile by an enzymatic process. It is, however, visaged that the use of a combination of an appropriate amount a CBD and an a-amylase will result in enhanced starch-removal rformance in the context of textile de-sizing relative to that hieved using an a-amylase in the absence of the CBD.
Yet another aspect of the invention relates to a method for covering starch from starch-containing corn kernels, wherein e kernels are steeped in a medium (normally predominantly ueous) comprising a CBD and a xylanase,
rbohydrate-binding domains
A carbohydrate-binding domain (CBD) is a polypeptide amino id 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 -I 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 I 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 pegracjation of Insoluble Carbphydrates. 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. Tonmie 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, therein, 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 se 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.
Bacterial. 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.
-ellulases fcellulase genes) useful for preparation of CBDs
Techniques suitable for isolating a cellulase gene are well Icnown 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 (recombinant) endogluc¬anases, 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 Pseudoraonas, Bacillus, Cellulomonas, Clostridium, Microspora, Thermotoga, Caldocellutn and Actinomycets such as Streptoiayces, Termomonospora and Acidothemus, in particular from the group consisting of Pseudomonas cellulolyticus. Bacillus lautus, Cellulomonas fimi, Clostridium thermoc&llum, in particular c. stercorarium, Microspora bispora, Termomonospora fusca, Termomonospora cellulolyticum and Acidothemns 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
Botrytls.
A preferred useful acid cellulase is one derived from or producible by fungi from the group of species consisting of Tri-choderma viride, Trichoderma reesei, Trichoderma longibrachiatum, Myrothecium verirucaria, 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 con¬sisting of Aspergillus, Penicillium, Myceliophthora, Humicola, Irpex, Fusarium, Stachybotrys, Scopulariopsis, Chaetomium, Myco-gone, Verticillium, Myrothecium, Papulospora, Gliocladium, Cepha-losporium and Acrejuonium.
A preferred alkaline cellulase is one derived from or produ¬cible by fungi from the group of species consisting of Humicola insolens, Fusarium oxysporum, Myceliopthora theirmophila^ Penicillium janthinellum and Ce^alosporium sp., preferably from the group of species consisting of Humicola insolens DSM 1800, Fusarium oxysporum DSM 2672, Myceliopthora thermophila CBS 117.65, and Cephalosporium 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 insolens DSM 1800, or which is a derivative of the latter "43kD endoglucanase
rpu
and exhibits cellulase activity (e.g. Carezyme ).
Other exeunples 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 fusarium.

other proteins fprotein genes) useful for preparation of CBDs
Examples of other types of hydrolytic enzymes which comprise 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 foiuid, 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 I be made to P. Tomme et al. [op cit.) for fvurther 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). f 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, arabinofiiranosidase genes, acetylesterase genes, chitinase genes (and other relevant genes) t 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.
f 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
k 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 inyention 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 a-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 a-amylases include, for example, a-amylases. obtainable from Bacillus species, in psurticular 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 Sovo Nordisk A/S, Bagsvaerd, Denmark) , and Rapidase™ and Maxamyl ?™ (available from Gist-Brocades, Holland).
starch- or starch-fraoment-debranching enzymes
[soamvlases: isoamylases (EC 3.2.1.68) appropriate for use in ;ombination with CBDs in the context of the present invention .nclude these of bacterial origin. Chemically or genetically mod-.fied mutants of such isoamylases are included in this ionnection. Relevant isoamylases include, for example, .soamylases obtainable from Pseudomonas species, (e.g. 'seudomonas sp. SMPl or P. amyloderomosa SB15), Bacillus species e.g. B. amyloliquefaciens), Flavobacteriwn species or Cytophaga Lysobacter) species.
ullulanases: pullulanases (EC 3.2.1.41) appropriate for use in ombination with CBDs in the context of the present invention nclude those of bacterial origin. Chemically or genetically mod-fied mutants of such pullulanases --^re included in this onnection. Relevant pullulanases include, for example,

pullulanases obtainable from Bacillus species (e.g. B. acidopullulyticus; such as Promozyme™, from Novo Nordisk A/S).
Other polvsaccharide-hydrolvsina enzymes 'Further enzymes of particular relevance for use in coabination with CBDs in the context of the present invention particularly in the cr xt of improving starch recovery from corn (maize) in corn-s'-e- ;ing processes (vide supra) - include xylanases, such as ty ~r classified under EC 3.2.1.8 or EC 3.2.1.32. Chemically or genetically modified mutants of xylanases are included in this connection. An example of a relevant xylanase is Shearzyme"* available from Novo Nordisk A/S, or Spezyme® CP available from Genencor, USA, a H. insolens xylanase (produced as described in Example 2 of WO 92/17573, 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), ' mase II (produced as described in WO 94/21785), a xylanase -reduced by B. pimilus strain DSM 6124 as described in WO 92/03540.
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 thermopbilus, Thermococcus lithoralis, Clostridium butyricum, streptococcus 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 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 fc 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 he produced, e.g., as disclosed in US 4,106,991 and US 4,661,452 (both to Novo Industri A/S), and may option¬ally be coated by methods known in the art.
The desirable levels of enzyme activity and the amount of 2BD, 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 ;ase of a liquefaction process) upon which the enzyme/CBD sombination is to act. The skilled person will be able to aetermine suitable dosages of enzyme activity and of CBD on the basis of methods known in the art.
Kateriala and Methods
Pullulanases: Proraozyme® (available from Novo Nordisk) derived from Bacillus acidopullulyticus (described in EP 63,909). Xyianase: Shearzyme^ (available from Novo Nordisk) Cellulase: Carezyme* (Novo Nordisk A/S)
Steepzyrae™ is an experimental muXtiactivity enzyme complex from Novo Nordisk produced from a selected sttain of Aspergillus. Steepzyme™ : enzyme preparation containing a number of the following activities: pectolytic, cellulolytic, and hemicellulolytic activities.
CBD: Cellulose-binding Domain di-mer derived from Clostridium stercorarium (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 stercorarium xynA gene encoding xyianase 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 xyianase A of Clostridium stercorarium) . Amylopectin (Waxy maize starch, cerestar)

Flour
The flovir used in the following Examples has the following coniponents :
i Fakta flour: a cominercial flour of non-specified type ("LuJcsus hvedemel", prepared by Dagligvaregruppen, DK-7100 Vejle).
Determination of a-amylolytic activity (KNU^
The a-amylolytic activity of an enzyme may be determined using
\ potato starch as substrate. This method is based on the break¬down (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
I 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 a-Amylase Unit (KNU) is defined as the amount of enzyme (activity) which, under standard conditions (i.e. at ) 37+0.05°C, 0.0003 M Ca^*, pH 5.6) dextrinizes 5.26 g starch dry substance (Merck Amylum solubile).
I

Determipation of pn^lulanase 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: ACC
pH: 5.0
Reaction time: 30 minutes
A detailed description of the analysis method (AF 190) is available on request.
3^telimination of xylanase activity (FXU^
The endo-xylanase activity is determined by an assay, in which ::he xylanase sample is incubated with a remazol-xylan substrate (4-0-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue i, Fluka), pH 6.0. The incubation is performed at 50"C for 30 nin. The background of non-degraded dyed siibstrate is precipi¬tated by ethanol. The remaining blue colour in the supernatant is aetermined spectrophotometrically at 585 nm 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.

petermination of endo-glucanase activity (EGUl
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. l 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 + epzyme combinations in starch processing
Test conditions (e.g. conditions of pH, temperature, calcium concentration etc.) suitable for testing, e.g., CBD + a-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 conteict.
Steeping method
steeping is carried out as indicated below and in Figure 3.
1. Steeping.
The corn dry substance and the starch content is Bieaaured 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 Naci solution and mixed. The volume after removal of the germs is about 600 ml.
4. Germ wash
The germ is washed with water on a vibrating screen (45 t^m) until wash vater stains yellow with iodine solution. The water then is recovered. The germs dry substance, amount, and starch content are measured.
5. 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.
5b. A FRYMA MZ-110 mill is used. Milling time 1 minute. Use of 1.5 litre slurry.
6. Fibre wash
Wash with water on vibrating screen (4 Spjn) until wash water stains yellow with iodine solution. Measurement of fibres dry substance, amount, and starch content.
7. Starch/Gluten isolation.
Collect slurry throughput from fibre wash on Btichner funnel placed on top of a vacuum flask where vacuum is applied. Measurement of starch/gluten dry substance, amount, and starch content. Wash water measurement of amount and dry substance. Mass balance on material dry substance basis. Mass balance on starch.
EXAMPLES
EXAMPLE 1
Dqbranchina of amylopectin with a debranchina enzyme and
Carez'
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 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. j
Except for 2 glass tubes (control tubes), a basic dosage of 2 PUN/g DS of debranching enzyme (Promozyme'™ 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:

protein/ml (e.p.= enzyme protein)
n) Gelatine, Merck 4078, diluted to 10 mg/ml
After addition of enzymes and gelatine the glass tubes were
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.
20 mg of each dried sample were dissolved in 8 ml DMSO
(dimethylsulfoxide) by heating to JO^C overnight in a water bath. The solutions were filtered through a l 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 30O 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 NaN03 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 (KW 180-1,600,000) from Polymer.
Labs. *
The experiments showed that a dosage of about 2 PUN/g DS 3f debranching enzyme give a degree of debranching of about 40-)0 %. This dosage of debranching enzyme was chosen so that it rave a reasonable degree of debranching and still allowed for iurther enhancement.
The degree of debranching is evaluated by GPC as described .nd taken as the weight fraction of material with a molecular eight below 20,000 Daltons. If 40% of the material have a olecular weight below 20,000 Daltons the degree of debranching s 40%.
Different dosages of gelatine (0.25, 0.5 and 0.75 mg/g DS s described) were incubated with debranching enzyme (2 PUN/g S) in order to see a possible effect of protein. In table l le degree of debranching is listed for the different dosages f gelatine.

sis dosage of debranching enzyme of 2 PUN/g DS.
From the results in Table 1 there appears to be no effect addition of gelatine during debranching of amylopectin with branching enzyme. Up to a gelatine dosage of 0.5 mg/g DS the gree of debranching is about S2 % and at 0.75 mg/g DS it is out 47 %. If gelatine has any effect on the efficiency of branching it appears to be negative at high dosages.
Table 2 show the results from debranching with 2 PUN/g DS debranching enzyme with different dosages of cellulase omprising a CBD).


The results in Table 2 are based on 4 repetitions. In the Figure l the results are showed graphically.
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 3ellulase/g DS the degree of debranching has increased to about 55 %, corresponding to an enhancement of the debranching afficiency of 38 %.
The cellulase used, i.e. Carezyme^, which contains a CBD, snhance the debranching efficiency of debranching enzyme. An equivalent dosage of gelatine, a protein without CBD, has no or 1 little negative effect on the debranching efficiency.
Ixample 2
)ebranchina of amylopectin with a debranching enzyme and a pure
ISg
jnylopectin (Waxy maize starch, Cerestar) was suspended in leionized water to a 7 % DS slurry by stirring for 20 minutes, .liquots of 20 g of the slurry was added to glass tubes with crew caps. The starch slurries in the glass tubes were heated o 140°c for 9 minutes in an oil bath. After the gelatinisation he starch solutions were air cooled to 60*0 (after about 10 inutes). After cooling the starch solutions were well shaken nd then transferred to a water bath heated to 60°C, ebranching enzyme(Promozyme 600 L, batch AGN 2008; diluted to 0 PUM/ml) and CBDs {Clostridium steacorarium, batch no. opr 51195, MB 206#9644, 4 mg/ml) were added to the starch.


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
aimethylsulfoxide (DMSO)by heating to 70°C overnight in a water oath. 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., JK, The samples were eluted with 99.9 % (v/v) DMSO and 0.1 % 0.Ol M NaN03 using a flow rate of 0.5 ml/minutes. The columns
were heated to 40 C. 100 ml samples were analysed.

The molecuiar weight distributions were calculated using a Millenniuma 2 010 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|) fExample 1).
In Table 3 the degree of debranching for different dosages of debranching enzyme is listed.

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.

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 l) 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 Clostridium steacorarium snhance the debranching efficiency of debranching enzyme.
eXAMPLE 3
discosity reduction by CBD and xylanase
The viscosity reduction provided by the combination CBD and cylanase 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: O.i protein per gram Dry Solids.
A sample comprising 7.5 FXU xylanase only and a blank sample is ased as control (no CBD and no xylanase added) are also tested.
The flour and water are stirred by hand for 30 seconds and then nixed 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 mea¬sured 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 evalu¬ated by a centrifugation test:

FaKta flour and water is mixed accprding to the procedure desc¬ribed in Example 3. After blending 10 ml of the batter is cen-trifugated (Megaf uge 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
method for steeping of starch-containing corn kernels is
described above in the "Steeping method" section and further in
figure 3. .
0.2% SteepzymeTM (based on corn dry substance) with and rithout CBD is added to the steep.
Simultaneous addition of Steepzyme™ and CBD give a higher tarch yields, less starch in germs and fibres, lower viscosity nd less sludge in the steepwater, less fibres in downstream peration, reduced steeping time compared to a steeping trials ith addition of Steepzyme ™ alone.
this application has been divided out of Indian Patent applicatlon No,2706/KA3/97 which relates to a method for producing liquified starch.

WE CLAIM:
1. A proceoss for producing dextrose from starch by saooharification comprising the steps of liquifying starch by known means and adding a pure carbohydrate binding donain (CBD) and atleast one anylopeotin-debranching enyme, to said liquified starch and recovering dextrose from the reaction mixture by method known per se.
2. The process as claimed in claim 1, wherein said auylopeotin-debranching enzyme is an isoanylase or a pullulanase.
3. The process as claimed in claim 1, wherein said amylopectln debranohlng enzyne is a gluooanylase.

4. The process as claimed in clains 1-3, wherein starch is liquified by treatment with an -amylase.
5. The process as claimed in claims 1-3, wherein starch is liquified by treatment with a combination of pure carbohydrate binding domain and atleast one amylolytlc enzyme between pH 5.5 and 8.2 at a temperature range of 95°C to 180°C.

6. A process for producing dextrose from staroh by saooharification substantially as herein described.