ES2366952T3 - PROCESS OF HYDROLYSIS OF ALMIDÓN. - Google Patents

Abstract

Process for producing a soluble starch hydrolyzate, the process comprising subjecting an aqueous suspension of granulated starch at a temperature below the initial gelatinization temperature of said granulated starch to the action of a first enzyme and a second enzyme, said First enzyme: (a) is an element of the 13 family of glycoside hydrolases; (b) has alpha-1,4-glycosidic hydrolysis activity, and; (c) comprises a functional carbohydrate binding module (CBM) of the CBM family 20, said CBM has an amino acid sequence selected from the group consisting of an amino acid sequence with at least 60% homology with SEQ ID NO: 1, an amino acid sequence with at least 75% homology with SEQ ID NO: 2, and an amino acid sequence with at least 90% homology with SEQ ID NO: 3; and said second enzyme is selected from the list comprising a fungal alpha-amylase (EC 3.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a process for the hydrolysis of granulated starch in a soluble starch hydrolyzate at a temperature below the initial gelatinization temperature of said granulated starch.

BACKGROUND OF THE INVENTION

[0002] A large number of processes have been described to convert starch into starch hydrolysates, such as maltose, glucose or specialty syrups, either for use as sweeteners or as precursors for other saccharides such as fructose. Glucose can also be fermented to ethanol or other fermentation products, such as citric acid, monosodium glutamate, gluconic acid, sodium gluconate, calcium gluconate, postasic gluconate, delta lactone glucono, sodium erythorbate, itaconic acid, lactic acid, gluconic acid; ketones; amino acids, glutamic acid (sodium monoglutaminate), penicillin, tetracycline; enzymes; vitamins, such as riboflavin, B12, beta-carotene or hormones.

[0003] Starch is a high molecular weight polymer consisting of chains of glucose units. It usually consists of approximately 80% amylopectin and 20% amylose. Amylopectin is a branched polysaccharide in which linear chains of alpha-1,4 D-glucose residues are linked by alpha-1,6 glycosidic bonds.

[0004] Amylose is a linear polysaccharide consisting of D-glucopyranoside units linked together by alpha-1,4 glycosidic bonds. In the case of converting the starch into a soluble starch hydrolyzate, the starch is depolymerized. The conventional depolymerization process consists of a gelatinization step and two consecutive process steps, that is, a liquefaction process and a saccharification process.

[0005] Granulated starch consists of microscopic granules, which are insoluble in water at room temperature. When an aqueous suspension of starch is heated, the granules swell and eventually burst, dispersing the starch molecules in the solution. During this "gelatinization" process there is a spectacular increase in viscosity. Since the level of solids is 30-40% in a typical industrial process, the starch has to be diluted or "liquefied" so that it can be handled. This reduction in viscosity is obtained today mainly by enzymatic degradation. During the liquefaction step, long chain starch is degraded into smaller branched and linear units (maltodextrins) by an alpha-amylase. The liquefaction process is typically carried out at about 105-110 ° C for about 5 to 10 minutes, followed by 1-2 hours at about 95 ° C. The temperature is then decreased to 60 ° C, a glucoamylase or a betaamylase and optionally a de-branching enzyme, such as an isoamylase or a pululanase, are added, and the saccharification process continues for approximately 24 to 72 hours.

[0006] From the previous explanation, it will be obvious that the conventional starch conversion process consumes a lot of energy due to the different temperature requirements during the different steps. Therefore, it is desirable to be able to select the enzymes used in the process so that the overall process can be performed without having to gelatinize the starch. Such processes are the subject of patents US4591560, US4727026 and US4009074 and EP0171218. WO 02/068589 discloses a thermostable alpha-amylase for starch hydrolysis.

[0007] The present invention relates to a one-step process for converting granulated starch into soluble starch hydrolyzate at a temperature below the initial gelatinization temperature of starch.

SUMMARY OF THE INVENTION

[0008] In a first aspect, the invention provides a process for producing a soluble starch hydrolyzate, the process comprising subjecting the aqueous suspension of granulated starch to a temperature below the initial gelatinization temperature of said granulated starch at the action of a first enzyme and a second enzyme, wherein said first enzyme; (a) is an element of the 13 family of glycoside hydrolases; (b) has alpha-1,4-glycosidic hydrolysis activity, and; (c) comprises a functional carbohydrate binding module (CBM) belonging to the family 20 of CBM, said CBM has an amino acid sequence selected from the group consisting of an amino acid sequence with at least 60% homology to the SEQ ID NO: 1, an amino acid sequence with at least 75% homology with SEQ ID NO: 2, and an amino acid sequence with at least 90% homology with SEQ ID NO: 3; and said second enzyme is selected from the list comprising a fungal alpha-amylase (EC 3.2.1.1), a beta-amylase (E.C. 3.2.1.2), and a glucoamylase (E.C.3.2.1.3). The process of the first aspect of the invention can be carried out as a one-step process and / or as a process comprising one or more steps.

[0009] In a second aspect, the invention provides a process for the production of fructose starch syrup (HFSS), the process including the steps of; (a) subjecting an aqueous suspension of granulated starch at a temperature below the initial gelatinization temperature of said granulated starch for the action of a first enzyme and a second enzyme, said first enzyme; (i) it is an element of the 13 family of glycoside hydrolases (ii) it has an alpha-1,4-glycosidic hydrolysis activity, and; (iii) comprises a functional carbohydrate binding module (CBM) of the CBM family 20, said CBM has an amino acid sequence selected from the group consisting of an amino acid sequence with at least 60% homology with SEQ ID NO: 1, an amino acid sequence with at least 75% homology with SEQ ID NO: 2, and an amino acid sequence with at least 90% homology with SEQ ID NO: 3 and said second enzyme is selected from the list comprising a fungal alpha-amylase (EC 3.2.1.1), a beta-amylase (EC 3.2.1.2), and a glucoamylase (EC3.2.1.3), to produce a soluble starch hydrolyzate, (b) submit said soluble starch hydrolyzate, for conversion into fructose-rich corn syrup (HFCS).

[0010] In a third aspect, the invention provides a process for the production of a fermentation product, the process including the steps of: (a) subjecting an aqueous suspension of granulated starch at a temperature below the initial gelatinization temperature of said granulated starch to the action of a first enzyme and a second enzyme, said first enzyme; (i) it is an element of the 13 family of glycoside hydrolases; (ii) has an alpha-1,4-glycosidic hydrolysis activity, and; (iii) comprises a functional carbohydrate binding module (CBM) of the CBM family 20, said CBM has an amino acid sequence selected from the group consisting of an amino acid sequence with at least 60% homology with SEQ ID NO: 1, an amino acid sequence with at least 75% homology with SEQ ID NO: 2, and an amino acid sequence with at least 90% homology with SEQ ID NO: 3; and said second enzyme is selected from the list comprising a fungal alpha-amylase (EC 3.2.1.1), a beta-amylase (EC 3.2.1.2), and a glucoamylase (EC3.2.1.3), to produce a soluble starch hydrolyzate , (b) subjecting said soluble starch hydrolyzate to fermentation in a fermentation product. In a preferred embodiment of the third aspect the fermentation product is ethanol.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0011] The term "granulated starch" is understood as untreated raw starch, that is, starch that has not been subjected to gelatinization. Starch is formed in plants as tiny water-insoluble granules. These granules are stored in starches at temperatures below the initial gelatinization temperature. When put in cold water, the grains may absorb a small amount of the liquid. Up to 50 ° C - 70 ° C the swelling is reversible, the degree of reversibility depends on the particular starch. With higher temperatures, an irreversible swelling called gelatinization begins.

[0012] The term "initial gelatinization temperature" is understood as the minimum temperature at which starch gelatinization begins. The starch heated in water begins to gelatinize between 50 ° C and 75 ° C; The exact temperature of gelatinization depends on the particular starch and can easily be determined by the person skilled in the art. Thus, the initial gelatinization temperature may vary according to the plant species, according to the particular variety of the plant species, as well as with the growing conditions. In the context of this invention the initial gelatinization temperature of a given starch is the temperature at which birefringence is lost in 5% of the starch granules using the method described by Gorinstein. S. and Lii. C., Starch / Stärke, Vol. 44 (12) pp. 461-466 (1992).

[0013] The term "soluble starch hydrolyzate" is understood as the soluble products of the processes of the invention and may comprise mono, di, and oligosaccharides, such as glucose, maltose, maltodextrins, cyclodextrins and any mixture thereof. Preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% of the dried solids of the granulated starch is converted into a soluble starch hydrolyzate.

[0014] The term "specialty syrups" is a term recognized in the art and is characterized by the equivalent of dextrose (DE) and the carbohydrate spectrum (see the article "New Specialty Glucose Syrups", p. 50, in the textbook "Molecular Structure and Function of Food Carbohydrate", edited by GG Birch and LF Green, Applied Science Publishers LTD., London). Typically specialty syrups have a DE in the range of 35 to 45.

[0015] "Family 13 of glycoside hydrolases" is defined in the context of this invention as the group of hydrolases comprising a catalytic module with a structure (beta / alpha) 8 or TIM barrel and acting on starch and related substrates through an alpha retention reaction mechanism (Koshland, 1953, Biol.Rev.Camp.Philos.Soc 28, 416-436).

[0016] Enzymes with "alpha-1,4-glucosidic hydrolysis activity" are defined in the context of this invention defined as the group of enzymes that catalyze the hydrolysis and / or synthesis of alpha-1,4-glucosidic bonds such and as defined by Takata (Takata et al, 1992, J. Biol. Chem. 267, 18447-18452) and by Koshland (Koshland, 1953, Biol. Rev. Camp. Philos. Soc 28, 416-436).

[0017] The "carbohydrate binding module of family 20" or a CBM-20 module is defined in the context of this invention as a sequence of approximately 100 amino acids with at least 45% homology to the binding module to Carbohydrates (CBM) of the polypeptide described in Figure 1 by Joergensen et al (1997) in Biotechnol.

Lett. 19: 1027-1031. The CBM comprises the last 102 amino acids of the polypeptide, that is, the sequence from amino acid 582 to amino acid 683. The numbering of glycoside hydrolase families applied in this description follows the concept of Coutinho, P.M. & Henrissat, B. (1999) CAZy -Carbohydrate-Active Enzymes at the URL: http://afmb.cnrsmrs.fr/~cazy/CAZY/index.html or alternatively Coutinho, P.M. & Henrissat, B. 1999; The modular structure of cellulases and other carbohydrate-active enzymes: an integrated database approach. In "Genetics, Biochemistry and Ecology of Cellulose Degradation"., K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T. Kimura eds., Uni Publishers Co., Tokyo, p. 15-23, and Bourne, Y. & Henrissat, B. 2001 Glycoside hydrolases and glycosyltransferases: families and functional modules, Current Opinion in Structural Biology 11: 593-600.

[0018] A carbohydrate-binding module (CBM) is an amino acid sequence of polypeptide that preferentially binds to a poly or oligosaccharide (carbohydrate), frequently - but not necessarily exclusively - to a water-insoluble form (including crystalline). of the same.

[0019] Although several types of CBMs have been described in the scientific and patent literature, most of them - many of which are derived from cellulolytic enzymes (cellulases) - are commonly referred to as "cellulose binding modules"; A typical cellulose binding module will then be a CBM that occurs in a cellulase. Also, other subclasses of CBMs would include, e.g. eg, chitin binding modules (CBMs that typically occur in chitinases), xylan binding modules (CBMs that typically occur in xylanases), mannan binding modules (CBMs that typically occur in mannanases), starch binding modules (CBMs that can occur in certain amylolytic enzymes, such as certain glucoamylases, or in enzymes such as cyclodextrin glucanotransferases), or in alpha-amylases.

[0020] CBMs are found as integral parts of large polypeptides or proteins that consist of two or more amino acid sequence regions of the polypeptide, especially hydrolytic enzymes (hydrolases) that typically comprise a catalytic module containing the active site for hydrolysis. of the substrate and a carbohydrate binding module (CBM) for the substrate binding of the carbohydrate in question. Such enzymes may comprise more than one catalytic module and one, two or three CBMs, and optionally further comprise one or more amino acid sequence regions of polypeptide that connect the CBM (s) with the catalytic module (s) ), a region of the last type normally referred to as "linker". Examples of hydrolytic enzymes that comprise a CBM - some of which have already been mentioned above - are cellulases, xylanases, mannases, arabinofuranosidases, acetylesterase and chitinases. CBMs have also been found in algae, e.g. eg, in the red algae Porphyra purpurea in the form of a non-hydrolytic polysaccharide binding protein.

[0021] In proteins / polypeptides where CBMs take place (eg, enzymes, typically hydrolytic enzymes), a CBM can be located in the N or C terminal or in an internal position.

[0022] This part of a polypeptide or protein (eg hydrolytic enzyme) that constitutes a CBM per se typically consists of more than about 30 and less than about 250 amino acid residues. For the invention, enzymes comprising a CBM with an amino acid sequence selected from the group consisting of an amino acid sequence with at least 60% homology with SEQ ID NO: 1, an amino acid sequence with at least 75 are preferred % homology with SEQ ID NO: 2, and an amino acid sequence with at least 90% homology with SEQ ID NO: 3

[0023] The "homology" of polypeptides referred to in this description is understood as the degree of identity between two sequences that indicate a derivation of the first sequence from the second. Homology can be adequately determined by computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA). 53711) (Needleman, SB and Wunsch, CD, (1970), Journal of Molecular Biology, 48, 443-453). The following settings are used to compare the amino acid sequence: GAP 3.0 creation penalty and GAP 0.1 extension penalty.

[0024] The enzyme to be used as the first enzyme of the present invention is a four-module alpha-amylase consisting of a nucleus of three amylase modules and a separate family 20 carbohydrate-binding module. The alpha-amylase may be a wild-type alpha-amylase derived from fungal or bacterial sources, or they may be mutants, modified variants of proteins, or other variants of said wild types, or they may be hybrids of wild-type variants or types.

[0025] Preferably alpha-amylase is a wild type enzyme. More preferably alpha-amylase is a variant and / or hybrid of the above alpha-amylases comprising amino acid modifications that lead to increased activity, increased protein stability at low pH, and / or high pH, increased stability towards depletion. of calcium, and / or increased stability at elevated temperature.

[0026] The term "enzymatic hybrids" referred to in this description is understood as modified enzymes comprising an amino acid sequence of an amylolytic enzyme [which in the context of the present invention may be, e.g. eg, an alpha-amylase (EC 3.2.1.1), an isoamylase (EC 3.2.1.68) or a pululanase (EC 3.2.1.41)] linked (i.e. covalently linked) to an amino acid sequence comprising a CBM The CBM is preferably, but not exclusively, fused to the N-terminal. The hybrid can comprise more than one CBM.

[0027] Enzymatic hybrids containing CBM, as well as detailed descriptions of the preparation and purification thereof, are known in the art [see, p. eg, WO 90/00609, WO 94/24158 and WO 95/16782, as well as Greenwood et al., Biotechnology and Bioengineering 44 (1994) p. 1295-1305]. These can be prepared, e.g. eg, transforming into a host cell a DNA construct comprising at least one DNA fragment encoding the cellulose binding module linked, with or without a linker, to a DNA sequence encoding the enzyme of interest, and culturing the host cell transformed to express the molten gene.

[0028] The construction of a hybrid protein between a carbohydrate-binding module (CBM) and an alpha-amylase requires one or more of the following steps to obtain a stable, expressible and applicable enzyme.

1) To identify possible crossing points it is often necessary to align the CBM donor molecule with the donor of the catalytic modules using conventional methods. If the homology is relatively high there may be several possible crossing points. However, if the homology is low or if the sequence of the catalytic module and the CBM are available, respectively, the CBM can be attached as a link to the catalytic module, either at the beginning of the sequence, that is, in the inserted N-terminal after a signal sequence at the end, or at the C-terminal before the termination signal. Regardless of whether the CBM is located in the N-terminal or in the C-terminal, it may be beneficial to eliminate a few amino acids or insert several amino acids as linkers to obtain an expressible and stable enzyme in use.

2) The construction of the hybrid DNA of the genes encoding the CBM and the amylolytic module according to the considerations made according to 1) can be carried out by methods known to those skilled in the art. These methods include, among others, PCR reactions using primers designed to hybridize on the resulting DNA crossing point, DNA digestion followed by in-vivo ligation or combination, for example, by yeast.

3) A simple fixation of a CBM to an amylolytic module often results in a hybrid protein that is poorly expressed due to problems of folding or stability or in a hybrid protein lacking sufficient stability and / or activity in a given application. To overcome such problems, the hybrid protein can be engineered by either directed mutagenesis methods or by more random methods. This includes both amino acids in the modules of the CBM and in the amylolytic modules, such as optimizing the transition from the amylolytic module to the CBM, with respect to the length and sequence of amino acids.

[0029] As a first enzyme for the present invention, hybrid enzymes comprising a CBM comprising an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO are preferred : 3, as well as enzymes with an amino acid sequence selected from the group consisting of an amino acid sequence with at least 60%, at least 65%, at least 70%, at least 75%, at least 80 %, at least 85%, at least 90%, at least 95%, at least 98%, such as at least 99% homology with SEQ ID NO: 1, an amino acid sequence with at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, such as at least 99% homology with SEQ ID NO: 2 , and an amino acid sequence with at least 90%, at least 95%, at least 98%, such as at least 99% homology with SEQ ID NO: 3

[0030] Hybrid enzymes comprising an amino acid sequence with alpha-amylolytic activity and comprising an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NO: 4, SEC are also preferred as the first enzyme for the present invention. ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO : 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18, as well as enzymes comprising an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, such as at least 99% homology with an amino acid sequence selected from the group consisting of amino acid sequences SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10 , SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

[0031] Preferably, the first enzyme of the present invention comprises a CBM and / or an alpha-amolytic sequence derived from fungi, such as from a strain of a species of Talaromyces, or from a strain of a species of Aspergillus such as A. awamori, A. kawachii, A. niger A. orizae, etc. or from a bacterium, such as from a strain of the Bacillus species, such as from a strain of B. amilolyquefaciens, B. flavothermus, B. licheniformis or B. stearothermophilus.

[0032] More preferred as the first enzyme of the present invention is a four-module alpha-amylase consisting of a three-module amylase core and a separate family-20 carbohydrate-binding module. Most preferred is a four module alpha-amylase comprising an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, such as at least 99% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22

[0033] Preferably the first enzyme of the present invention is a four-module alpha-amylase isolated from a fungus or a bacterium, such as from a Bacillus species, such as the polypeptides shown in SEQ ID NO: 20, and in SEQ ID NO: 21, or a strain of Bacillus flavothermus, such as the polypeptide shown in SEQ ID NO: 19, or a strain of Aspergillus kawachii such as the polypeptide shown in SEQ ID NO: 22.

[0034] More preferred as the first enzyme of the present invention is an alpha-amylase comprising an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, such as at least 99% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

[0035] The above alpha-amylases may be added in an amount of 0.001-1.0 KNU / g DS, preferably 0.002-0.5 KNU / g DS, preferably 0.02-0.1 KNU / g of DS.

Fungal alpha-amylase

[0036] A particular enzyme to be used as a second enzyme in the processes of the invention is a fungal alpha-amylase (EC 3.2.1.1), such as a Fungamyl-type alpha-amylase. In the present disclosure, the term "fungamyl type alpha-amylase" indicates an alpha-amylase that shows high homology, that is, more than 50%, 55%, 60%, 65%, 70%, 75%, 80 %, 85% or even 90% homology with the amino acid sequence shown in SEQ ID NO: 10 in WO96 / 23874. Fungal alpha-amylases may be added in an amount of 0.001-1.0 AFAU / g DS, preferably 0.002-0.5 AFAU / g DS, preferably 0.02-0.1 AFAU / g DS.

Beta-amylase

[0037] Another particular enzyme to be used as a second enzyme in the processes of the invention may be a beta-amylase.

(E.C 3.2.1.2). Beta-amylase is the name generally given to exoactive maltogenic amylases, which catalyze the hydrolysis of alpha 1,4 glycoside bonds in amylose, amylopectin and related glucose polymers, thus releasing maltose.

[0038] Beta-amylases have been isolated from various plants and microorganisms (W.M. Fogarty and C.T. Kelly, Progress in Industrial Microbiology, vol. 15, pp. 112-115, 1979). These beta-amylases are characterized by having optimum temperatures in the range of 40 ° C to 65 ° C and an optimum pH in the range of 4.5 to 7.0. Beta-amylases contemplated include Spezyme® BBA 1500 barley beta-amylase, Spezyme® DBA and Optima ™ ME, Optimalt ™ BBA from Genencor Int., As well as Novozym ™ WBA from Novozymes A / S.

Glucoamylase

[0039] Another particular enzyme to be used as a second enzyme in the processes of the invention may also be a glucoamylase (E.C.3.2.1.3) derived from a microorganism or a plant. Bacterial or fungal glucoamylases selected from the group consisting of Aspergillus glucoamylases, in particular G1 or G2 glucoamylase from A. niger (Boel et al. (1984), EMBO J. 3 (5), p. 1097 are preferred -1102), or variants thereof, such as those described in WO92 / 00381 and WO00 / 04136; A. awamori glucoamylase (WO84 / 02921), A. oryzae (Agric. Biol. Chem. (1991), 55 (4), pp. 941-949), or variants or fragments thereof.

[0040] Other variants of Aspergillus glucoamylase contemplated include variants to improve thermostability: G137A and G139A (Chen et al. (1996), Prot. Engng. 9, 499-505); D257E and D293E / Q (Chen et al. (1995), Prot. Engng. 8, 575-582); N182 (Chen et al. (1994), Biochem. J. 301, 275-281); disulfide bonds, A246C (Fierobe et al. (1996), Biochemistry, 35, 8698-8704; and introduction to proline residues in A435 and S436 (Li et al. (1997), Protein Engng. 10, 1199-1204 Other contemplated glucoamylases include Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO99 / 28448), Talaromyces leycettanus (U.S. Patent No. Re.32, 153), Talaromyces duponti, Talaromyces thermophilus (U.S. Patent No. 4,587,215) Bacterial glucoamylases contemplated include glucoamylases of the genus of Clostridium, in particular C. thermoamylolyticum (EP135.138), and C. thermohydrosulfuricum (WO86 / 01831) Preferred glucoamylases include glucoamylases derived from Aspergillus oryzae, such as a 50% glucoamylase with a , 55%, 60%, 65%, 70%, 75%, 80%, 85% or even 90% homology with the amino acid sequence shown in SEQ ID NO: 2 in WO00 / 04136. commercial products AMG 200L; AMG 300 L; SA N ™ SUPER and AMG ™ E (from Novozymes); OPTIDEX ™ 300 (from Genencor Int.); AMIGASE ™ and AMIGASE ™ PLUS (from DSM); G-ZYME ™ G900 (from Enzyme Bio-Systems); G-ZYME ™ G990 ZR (A. niger glucoamylase and low protease content).

[0041] Glucoamylases can be added in an amount of 0.02-2.0 AGU / g of DS, preferably 0.1-1.0 AGU / g of DS, such as 0.2 AGU / g of DS.

Additional enzymes

[0042] The processes of the invention can be carried out in the presence of a third enzyme. A third enzyme in particular may be a Bacillus alpha-amylase (often referred to as "Termamyl-type alpha-amylases"). Well-known Termamyl alphaamylases include alpha-amylase derived from a strain of B. licheniformis (commercially available as Termamyl), B. amyloliquefaciens, and B. stearothermophilus alpha-amylase. Other Termamyl type alpha-amylases include alpha-amylase derived from a strain of the Bacillus species NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, which are all described in detail in WO95 / 26397, and the alpha-amylase described by Tsukamoto et al., Biophysical Research Communications, 151 (1988), p. 25-31. In the context of the present invention a Termamyl type alphaamylase is an alpha-amylase as defined in WO99 / 19467 on page 3, line 18 to page 6, line 27. Variants and hybrids contemplated are described in WO96 / 23874 , WO97 / 41213 and WO99 / 19467. Specifically contemplated is a recombinant variant of B. stearothermophilus alpha-amylase with the mutations: I181 * + G182 * + N193F. Bacillus alpha-amylases can be added in effective amounts well known to those skilled in the art.

[0043] Another third enzyme in the particular process may be a de-branching enzyme, such as an isoamylase.

(E.C. 3.2.1.68) or a pululanase (E.C. 3.2.1.41). Isoamylase hydrolyzes the branched alpha-1,6-D-glucosidic linkages in amylopectin and beta-dextrin boundary and can be distinguished from pululanases by the inability of isoamylase to attack the pululane, and by the limited action at boundary alpha-dextrins . The de-branching enzyme can be added in effective amounts well known to those skilled in the art.

Embodiments of the invention

[0044] The starch suspension to be subjected to the processes of the invention may have 20-55% dry solids of granulated starch, preferably 25-40% dry solids of granulated starch, more preferably 30-35% of dried solids of granulated starch.

[0045] After being subjected to the process of the first aspect of the invention, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or preferably at least 99% of the dried solids of the granulated starch is converted into a soluble starch hydrolyzate.

[0046] According to the invention, the processes of the first and second aspects are carried out at a temperature below the initial gelatinization temperature. Preferably the temperature at which the processes are performed is at least 30 ° C, at least 31 ° C, at least 32 ° C, at least 33 ° C, at least 34 ° C, at least 35 ° C, at least 36 ° C, at least 37 ° C, at least 38 ° C, at least 39 ° C, at least 40 ° C, at least 41 ° C, at least 42 ° C, at least 43 ° C, at least 44 ° C, at least 45 ° C, at least 46 ° C, at least 47 ° C, at least 48 ° C, at least 49 ° C, at least 50 ° C, at least 51 ° C, at least 52 ° C, at least 53 ° C, at least 54 ° C, at least 55 ° C, at least 56 ° C, at least 57 ° C, at least 58 ° C, at least 59 ° C, or preferably at least 60 ° C.

[0047] The pH with which the process of the first aspect of the invention is carried out may be in the range of 3.0 to 7.0, preferably 3.5 to 6.0, or more preferably 4.0-5.0.

[0048] The exact composition of the products of the process of the first aspect of the invention, the soluble starch hydrolyzate, depends on the combination of enzymes applied, as well as the type of granulated starch processed. Preferably the soluble hydrolyzate is maltose with a purity of at least 85%, at least 90%, at least 95.0%, at least 95.5%, at least 96.0%, at least 96 , 5%, at least 97.0%, at least 97.5%, at least 98.0%, at least 98.5%, at least 99.0% or at least 99.5 %. Even more preferably the soluble starch hydrolyzate is glucose, and most preferably the starch hydrolyzate has a DX (percentage of glucose of the total solubilized dry solids) of at least 94.5%, at least 95.0 %, at least 95.5%, at least 96.0%, at least 96.5%, at least 97.0%, at least 97.5%, at least 98.0%, at least 98.5%, at least 99.0% or at least 99.5%. However, equally contemplated is the process where the product of the process of the invention, the soluble starch hydrolyzate, is a specialty syrup, such as a specialty syrup with a mixture of glucose, maltose, DP3 and DPn for use in The production of ice cream, cakes, candies, canned fruit.

[0049] The granulated starch to be processed in the processes of the invention can be obtained in particular from tubers, roots, stems, legumes, cereals or whole grain. More specifically, granulated starch can be obtained from grains, corn grits, cobs, wheat, barley, rye, sorghum, sago, cassava, tapioca, sorghum, rice, peas, beans, bananas or potatoes. Especially contemplated are both waxy and non-waxy types of corn and barley. The granulated starch to be processed may be of a highly refined starch quality, preferably with at least 90%, at least 95%, at least 97% or at least 99.5% purity or it may be one more starch crude containing material comprising whole ground grain including non-starchy fractions such as germ and fiber residues. The raw material, such as whole grain, is ground to discover the structure and allow further processing. Two milling processes are preferred according to the invention: wet and dry milling. In dry grinding, the whole hazelnut is ground and used. Wet milling provides a good separation of germ and flour (starch and protein granules) and is applied with some exceptions in places where starch hydrolyzate is used in syrup production. Both dry and wet mills are well known in the art of starch treatment and are also contemplated for the processes of the invention. The process of the first aspect of the invention can be carried out in an ultrafiltration system where the concentrate is kept in recirculation in the presence of enzymes, raw starch and water, and where the concentrate is the soluble starch hydrolyzate. Equally contemplated is the process carried out in a continuous membrane reactor with ultrafiltration membranes and where the concentrate is kept in recirculation in the presence of enzymes, raw starch and water, and where the permeate is the soluble starch hydrolyzate. The process carried out in a continuous membrane reactor with microfiltration membranes is also contemplated and where the concentrate is kept in recirculation in the presence of enzymes, raw starch and water, and where the permeate is the hydrolyzate of soluble starch.

[0050] In the process of the second aspect of the invention, the soluble starch hydrolyzate of the process of the first aspect of the invention is subjected to a conversion into syrup based on fructose-rich starch (HFSS), such as rich corn syrup in fructose (HFCS). This conversion is preferably achieved using a glucose isomerase, and more preferably by an immobilized glucose isomerase supported on a solid support. Isomerases contemplated include the commercial products Sweetzyme ™ IT from Novozymes A / S, G -zyme ™ IMGI and G-zyme ™ G993, Ketomax ™ and G-zyme ™ G993 from Rhodia, G-zyme ™ G993 liquid and GenSweet ™ IGI from Genencor Int.

[0051] In the process of the third aspect of the invention, the soluble starch hydrolyzate of the process of the first aspect of the invention is used for the production of fuel or potable ethanol. In the process of the third aspect, fermentation can be carried out simultaneously or separately / sequentially to the hydrolysis of the granulated starch suspension. When the fermentation is carried out simultaneously with the hydrolysis, the temperature is preferably between 30 ° C and 35 ° C, and more preferably between 31 ° C and 34 ° C. The process of the third aspect of the invention can be carried out in an ultrafiltration system where the concentrate is kept in recirculation in the presence of enzymes, raw starch, yeast, yeast nutrients and water, and where the permeate is a liquid containing ethanol. The process carried out in a continuous membrane reactor with ultrafiltration membranes is also contemplated and where the concentrate is kept in recirculation in the presence of enzymes, raw starch, yeast, yeast nutrients and water, and where the permeate is a liquid that ethanol.

[0052] The soluble starch hydrolyzate of the process of the first aspect of the invention can also be used for the production of a fermentation product comprising the fermentation of treated starch in a fermentation product, such as citric acid, monosodium glutamate, acid gluconic, sodium gluconate, calcium gluconate, potassium gluconate, glucono-delta-lactone, or sodium erythorbate.

[0053] In another embodiment the starch suspension is in contact with a polypeptide comprising a CBM, but no amylolytic module, that is, application of loose CBMs. The loose CBMs can be starch junction modules, cellulose junction modules, chitin junction modules, xylan junction modules, mannan junction modules, and other junction modules. Preferred CBMs in the present context are microbial CBMs, particularly fungal or bacterial CBMs. Particularly preferred are the starch binding modules shown in the present disclosure as the polypeptide sequences SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 or the starch binding modules described in provisional patent application American No. 60/511044 as SEQ ID NO: 12; the glucoamylase CBM of the Hormoconis species such as Hormoconis resine (Sin. Creosote fungus

or Amorphoteca resine) (SWISSPROT: Q03045), SEQ ID NO: 13; the CBM of Lentinula species such as Lentinula edodes (shiitake fungus) (SPTREMBL: Q9P4C5), SEQ ID NO: 14 the CBM of Neurospora species such as Neurospora crassa (SWISSPROT: P14804), SEQ ID NO: 15; CBM of Talaromyces species such as Talaromyces byssochlamydioides, SEQ ID NO: 16; the CBM of Geosmithia species such as Geosmithia cylindrospora, SEQ ID NO: 17; the CBM of Scorias species such as Scorias spongiosa, SEQ ID NO: 18; CBM of Eupenicillium species such as Eupenicillium ludwigii, SEQ ID NO: 19; the CBM of Aspergillus species such as Aspergillus japonicus, SEQ ID NO: 20; the CBM of Penicillium species such as Penicillium cf. miczynskii, SEQ ID NO: 21; CBM of Mz1 Penicillium species, SEQ ID NO: 22; the CBM of Thysanophora species, SEQ ID NO: 23; the CBM of Humicola species such as Humicola grisea var. thermoidea Many preferred CBMs include the CBMs described in provisional US Patent Application No. 60/511044 as SEQ ID NO: 24; the glucoamylase CBM of Aspergillus species such as Aspergillus niger, and as SEQ ID NO: 25; the glucoamylase CBM of Athelia species such as Athelia rolfsii. Also preferred for the invention is the application of any CBM having at least 50%, 60%, 70%, 80% or even at least 90% homology with any of the above-mentioned CBM amino acid sequences.

[0054] Loose CBMs can be applied to the granulated starch suspension in effective amounts.

MATERIALS AND METHODS

Alpha-Amylase Activity (KNU)

[0055] The amylolytic activity can be determined using potato starch as a substrate. This method is based on the decomposition of the enzyme modified potato starch, and the reaction is followed by samples of mixing the starch / enzyme solution with an iodine solution. Initially, a blackish blue color is formed, but during the decomposition of the starch the blue color weakens and gradually becomes a reddish brown, which is comparable to a standard of colored glass.

[0056] A Kilo Novo (KNU) unit of alpha-amylase is defined as the amount of enzyme that, under standard conditions (ie at 37 ° C +/- 0.05; 0.0003 M Ca2 +; and pH 5.6 ) dextrinizes 5.26 g of dry substance from soluble Merck Amylum starch.

[0057] A prospectus AF 9/6 describing this analytical method in more detail is available upon request to Novozymes A / S, Denmark, said prospectus is included herein by reference.

Glucoamylase Activity (AGU)

[0058] The Novo glucoamylase unit (AGU) is defined as the amount of enzyme that hydrolyzes 1 micromol of maltose per minute at 37 ° C and pH 4.3.

[0059] Activity is determined as AGU / ml by a modified method (AEL-SM-0131, available upon request in Novozymes) after using a GOD-Perid glucose diagnostic kit from Boehringer Mannheim, 124036. Standard: AMG standard, lot 7- 1195, 195 AGU / ml. 375 microL of substrate (1% maltose in 50 mM sodium acetate, pH 4.3) are incubated for 5 minutes at 37 ° C. 25 microL of enzyme diluted in sodium acetate are added. After 10 minutes, the reaction is stopped by adding 100 microL 0.25 M NaOH. 20 microL are transferred to a 96-well microtiter plate and 200 microL of GOD-Perid solution (124036, Boehringer Mannheim) is added. After 30 minutes at room temperature, the absorbency is measured at 650 nm and the activity is calculated in AGU / ml of the AMG standard. A leaflet (AEL-SM-0131) describing this analytical method in more detail is available upon request in Novozymes A / S, Denmark, which leaflet is included herein by reference.

Fungal alpha-amylase activity (FAU)

[0060] Fungal alpha-amylase activity can be measured in FAU (fungal alpha-amylase units). One (1) FAU is the amount of enzyme that under standard conditions (i.e. at 37 ° C and pH 4.7) decomposes 5260 mg of solid starch (soluble starch, Merck) per hour. An AF 9.1 / 3 leaflet, which describes this FAU test in more detail, is available upon request in Novozymes A / S, Denmark, which leaflet is included in this document by reference.

Acid alpha-amylase activity (AFAU)

[0061] The activity of acid alpha-amylase can be measured in AFAU (fungal acid alpha-amylase units), which are determined in relation to an enzymatic standard.

[0062] The standard used is AMG 300 L (from Novozymes A / S, wild-type Aspergillus niger G1 glucoamylase, also described in Boel et al. (1984), EMBO J. 3 (5), pp. 1097-1102 and in WO92 / 00381). The neutral alpha-amylase in this AMG decreases, after storage at room temperature for 3 weeks, of approx. 1 FAU / mL at less than 0.05 FAU / mL.

[0063] The acid alpha-amylase activity in this AMG standard is determined according to the following description. In this method 1 AFAU is defined as the amount of enzyme that degrades 5.26 mg of dried starch solids per hour under standard conditions.

[0064] Iodine forms a blue complex with starch, but not with its degradation products. Therefore, the intensity of the color is directly proportional to the concentration of starch. Amylase activity is determined using inverse colorimetry as a reduction in starch concentration under specific analytical conditions.

image 1

5

10

fifteen

twenty

25

30

Standard conditions / reaction conditions: (per minute)

Substrate: Starch, approx. 0.17 g / L Buffer: Citate, approx. 0.03 M Iodine (I2): 0.03 g / L CaCl2: 1.85 mM pH: 2.50 - 0.05 40 ° C temperature

incubation: Reaction time: 23 seconds Wavelength: lambda = 590nm 0.025 AFAU / mL concentration

enzymatic: Working range 0.01-0.04 AFAU / mL enzymatic:

[0065] If more detailed information is preferred, it can be found in EB-SM-0259.02 / 01, available upon request in Novozymes A / S, and incorporated by reference.

Beta-amvlase activity (DP °)

[0066] The activity of SPEZYME® BBA 1500 is expressed in degrees of diastatic power (DP °). It is the amount of enzyme contained in 0.1 ml of a 5% solution of the sample enzyme preparation that will produce sufficient reduction sugars to reduce 5 ml of Fehling solution when the sample is incubated with 100 ml of substrate for 1 hour at 20 ° C.

Pululanase activity (new Novo pululanase unit (NPUN)

[0067] Pululanase activity can be determined in relation to a pululane substrate. Pululane is a linear D-glucose polymer consisting essentially of maltotriosyl units linked by alpha 1.6 bonds. Endopululanases hydrolyse the alpha 1,6 bonds randomly, releasing maltotriose release, 63-alpha-maltotriosylmaltotriose, 63-alpha- (63-alpha-maltotriosyl-maltotriosyl) -maltotriose.

[0068] A new Novo pululanase unit (NPUN) is a unit of endo-pululanase activity and is measured in relation to a Novozymes A / S Promozyme D standard. Standard conditions are 30 minutes of reaction time at 40 ° C and pH 4.5 and with 0.7% of pululane as substrate. The amount of degradation product of red substrate is measured spectrophotometrically at 510 nm and is proportional to the endo-pululanase activity in the sample. A leaflet (EB-SM.0420.02 / 01) describing this analytical method in more detail is available upon request in Novozymes A / S, Denmark, which leaflet is included in this document by reference.

[0069] Under standard conditions an NPUN is approximately equal to the amount of enzyme that releases reducing carbohydrate with a reducing potency equivalent to 2.86 micromoles of glucose per minute.

Determination of sugar profile and solubilized dry solids

[0070] The sugar composition of the starch hydrolysates was determined by HPLC and subsequently the glucose yield was calculated as DX. BRIX, solubilized (soluble) dried solids of starch hydrolyzate were determined by measuring the refractive index.

materials

[0071] The following enzymatic activities were used. A bacterial alpha-amylase with a CBD with the sequence depicted in SEQ ID NO: 19 and the same bacterial alpha-amylase, but without the CBD module (SEQ ID NO: 4). A glucoamylase derived from Aspergillus niger with the amino acid sequence shown in WO00 / 04136 as SEQ ID NO: 2 or one of the variants described. A fungal acid alpha-amylase derived from Aspergillus niger.

[0072] Wheat starch (S-5127) was obtained from Sigma-Aldrich.

Example 1

[0073] This example illustrates the conversion of granulated wheat starch into glucose using a four-module bacterial alpha-amylase and a glucoamylase and a fungal acid amylase. A suspension with 33% dry solids (DS) of granulated starch was prepared by adding 247.5 g of wheat starch, while stirring, to 502.5 ml of water. The pH was adjusted with HCl to 4.5. The granulated starch suspension was distributed in 100 ml blue cap flasks with 75 g in each flask. The flasks were incubated with magnetic stirring in a 60 ° C water bath. At zero hours the enzymatic activities shown in Table 1 were dosed into the flasks. Samples were removed after 24, 48, 72, and 96 hours.

Table 1. The levels of enzyme activity used.

Bacterial alpha-amylase KNU / kg of DS Glucoamylase AGU / kg of DS Fungal acid alpha-amylase AFAU / kg of DS

100.0 200 50

[0074] The total dry solids of starch was determined using the following method. The starch was completely

10 hydrolyzed by adding an excess amount of alpha-amylase (300 KNU / Kg of dry solids) and placing the sample in an oil bath at 95 ° C for 45 minutes. Subsequently the samples were cooled to 60 ° C and an excess amount of glucoamylase (600 AGU / kg of DS) was added followed by an incubation for 2 hours at 60 ° C.

[0075] Dry solids soluble in the starch hydrolyzate were determined by measuring the refractive index in samples after filtering through a 0.22 microM filter. The sugar profile was determined

15 by HPLC. The amount of glucose was calculated as DX. The results are shown in Table 2 and 3.

Table 2. Soluble dry solids as a percentage of total dry substance at 100 KNU / kg of alpha dosage of alphaamylase.

KNU / kg of DS 24 hours 48 hours 72 hours 96 hours

100.0 92.5 96 97.3 99.2

Table 3. DX of the soluble hydrolyzate at 100 KNU / kg of DS of alpha-amylase dosage.

KNU / kg of DS 24 hours 48 hours 72 hours 96 hours

100.0 88.4 92.4 93.7 95.3

Example 2

[0076] This example illustrates the only partial conversion of granulated starch into glucose using a glucoamylase and a fungal acid alpha-amylase.

[0077] Flasks with 33% DS of granulated starch were prepared and incubated as described in example 1. At zero hours the enzymatic activities shown in Table 4 were dosed into the flasks. Samples were removed after 24, 48, 72, and 96 hours. The samples were analyzed as described in example 1. The results are shown in Table 5 and 6.

30 Table 4. The level of enzyme activity used.

Glucoamylase and AGU / kg of DS Fungal acid alpha-amylase AFAU / kg of DS

200 50

Table 5. Soluble dry solids as a percentage of total dry substance.

24 hours 48 hours 72 hours 96 hours

28.5 36.3 41.6 45.7 Table 6. DX of the soluble hydrolyzate.

24 hours 48 hours 72 hours 96 hours

27.7 34.9 39.2 42.2

5 Example 3

[0078] In Example 3, the conversion of granulated wheat starch to glucose was performed using a glucoamylase (200 AGU / kg of DS), a fungal acid amylase (50 AFAU / kg of DS) and bacterial alpha-amylase of four intact modules (SEQ ID NO: 19), also used in example 1 or the same four-module bacterial alpha-amylase, but without the CBD module (SEQ ID NO: 4) (100 KNU / kg DS). A suspension with 33% dry solids (DS) starch

The granulate was prepared and incubated as described in example 1. The samples were removed after 24, 46, 70, and 90 hours.

[0079] The total dry starch solids were determined as described in example 1. Dry solids soluble in the starch hydrolyzate and the sugar profile were determined as described in example 1. The results are shown in the table. 7 and 8.

15 Table 7. Soluble dry solids as a percentage of total dry substance. Enzymes: glucoamylase, fungal acid amylase and bacterial alpha-amylase with the CBD module (SEQ ID NO: 19) or without the CBD module (SEQ ID NO: 4).

24 hours 46 hours 70 hours 90 hours

Without CBD 89.7 92.4 92.4 92.5 With CBD 94.1 95.2 96.9 97.1

Table 8. DX of the soluble hydrolyzate: enzymes: glucoamylase, fungal acid amylase and bacterial alpha-amylase with the CBD module (SEQ ID NO: 19) or without the CBD module (SEQ ID NO: 4).

twenty

Without CBD With CBD 24 hours 85.9 89.9 46 hours 88.7 93.3 70 hours 89.0 93.0 90 hours 89.0 93.2

LIST OF SEQUENCES [0080]

25

<110> Novozymes A / S

<120> Cold liquefaction with four-domain alpha-amylase

30

<130> 10473 <160> 22

<170> PatentIn version 3.2

<210> 1

<211> 102

<212> PRT

<213> Bacillus flavothermus

<400> 1

image 1

<210> 2

<211> 99 10 <212> PRT

<213> Bacillus sp.

<400> 2

image 1

<210> 3

<211> 102

<212> PRT

<213> Alkaryl Bacillus

<400> 3

image 1

<210> 4

<211> 484

<212> PRT

<213> Bacillus flavothermus

<400> 4

image 1

image 1

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<210> 5

<211> 485

<212> PRT

<213> Bacillus sp

<400> 5

image 1

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<210> 6

<211> 484

<212> PRT

<213> alkaryl bacillus

<400> 6

image 1

image 1

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<210> 7

<211> 517

<212> PRT

<213> Bacillus sp .:

<400> 7

image 1

image 1

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<210> 8

<211> 550

<212>

PRT 5 <213> Unknown

<220>

<223> Unknown source

<210> 9

<211> 482

<212> PRT

<213> Unknown

<220>

<223> unknown source

<400> 9

<210> 10

<211> 482

<212> PRT

<213> Unknown

<220>

<223> Unknown source

<400> 10

<210> 11

<211> 482

<212> PRT

<213> Unknown

<220>

<223> Unknown source

<400> 11

<210> 12

<211> 482

<212>

PRT 5 <213> Unknown

<220>

<223> Unknown source

<210> 13

<211> 483

<212> PRT

<213> Bacillus licheniformis

<400> 13

<210> 14

<211> 483

<212> PRT

<213> Bacillus amyloliquefaciens

<400> 14

<210> 15

<211> 483

<212> PRT

<213> Bacillus stearothermophilus

<400> 15

<210> 16

<211> 485

<212>

PRT 5 <213> unknown

<220>

<223> unknown source

<210> 17

<211> 484

<212>

PRT 5 <213> Unknown

<220>

<223> unknown source

<210> 18

<211> 485

<212>

PRT 5 <213> unknown

10 <400> 8

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10 <400> 12

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10 <400> 16

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10 <400> 17

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<220>

<223> unknown source

10 <400> 18

image 1

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<210> 19

<211> 619

<212> PRT

<213> Bacillus flavothermus

<400> 19

image 1

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<210> 20

<211> 613

<212> PRT

<213> Bacillus

<400> 20

image 1

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<210> 21

<211> 619

<212> PRT

<213> Alkaryl Bacillus

<400> 21

image 1

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<210> 22

<211> 640

<212> PRT

<213> Aspergillus kawachii

<400> 22

image 1

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Claims (21)

1. Process for producing a soluble starch hydrolyzate, the process comprising subjecting an aqueous suspension of granulated starch at a temperature below the initial gelatinization temperature of said granulated starch to the action of a first enzyme and a second enzyme , said first enzyme:

(to)

it is an element of the 13 family of glycoside hydrolases;

(b)

It has alpha-1,4-glycosidic hydrolysis activity, and;

(C)

comprising a functional carbohydrate binding module (CBM) of the family 20 of CBM, said CBM has an amino acid sequence selected from the group consisting of an amino acid sequence with at least 60% homology with SEQ ID NO: 1 , an amino acid sequence with at least 75% homology with SEQ ID NO: 2, and an amino acid sequence with at least 90% homology with SEQ ID NO: 3;

and said second enzyme is selected from the list comprising a fungal alpha-amylase (EC 3.2.1.1), a betaamylase (E.C. 3.2.1.2), and a glucoamylase (E.C.3.2.1.3).

2.

Process according to claim 1, wherein the first enzyme comprises an amino acid sequence with at least 75% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18.

3.

Process according to any of the preceding claims, wherein the first enzyme comprises an amino acid sequence with at least 75% homology with an amino acid sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22.

Four.

Process according to any of the preceding claims, wherein the starch suspension has 20-55% dry solids of granulated starch.

5.

Process according to any of the preceding claims, wherein at least 85% of the dried solids of the granulated starch are converted into a soluble starch hydrolyzate.

6.

Process according to any of the preceding claims, comprising subjecting the suspension of granulated starch to the action of an isoamylase and / or a pululanase.

7.

Process according to any of the preceding claims, wherein the temperature is at least 58 ° C.

8.

Process according to any of the preceding claims, wherein the pH is in the range of 3.0 to

7.0.

9.

Process according to any of the preceding claims, wherein the percentage of glucose of the total solubilized dry solids of the soluble starch hydrolyzate is at least 94.5%.

10.

Process according to any of the preceding claims, wherein the granulated starch is obtained from tubers, roots, stems, or whole grain.

eleven.

Process according to any of the preceding claims, wherein the granulated starch is obtained from cereals.

12.

Process according to any of the preceding claims, wherein the granulated starch is obtained from corn, ears, wheat, barley, rye, millet, sago, cassava, tapioca, sorghum, rice or potatoes.

13.

Process according to any of the preceding claims, wherein the granulated starch is obtained from dry milling of whole grain or from wet milling of whole grain or from ground corn grits.

14.

Process according to any of the preceding claims, wherein the process is carried out in an ultrafiltration system and where the concentrate is kept in recirculation in the presence of enzymes, raw starch and water and where the permeate is the soluble starch hydrolyzate.

fifteen.

Process according to any of the preceding claims, wherein the process is carried out in a continuous membrane reactor with ultrafiltration membranes and where the concentrate is kept in recirculation in the presence of enzymes, crude starch and water and where the permeate is the hydrolyzate of soluble starch.

16.

Process according to any of the preceding claims, wherein the process is carried out in a continuous membrane reactor with microfiltration membranes and where the concentrate is kept in recirculation in the presence of enzymes, crude starch and water and where the permeate is the hydrolyzate of soluble starch.

17.

Process for the production of syrup based on fructose starch (HFSS), including the process the steps of:

a) subjecting an aqueous suspension of granulated starch at a temperature below the initial gelatinization temperature of said granulated starch to the action of a first enzyme and a second enzyme, said first enzyme:

(i)

 it is an element of the 13 family of glycoside hydrolases;

(ii)

 it has alpha-1,4-glycosidic hydrolysis activity, and;

(iii) comprises a functional carbohydrate binding module (CBM) of the CBM family 20, said CBM has an amino acid sequence selected from the group consisting of an amino acid sequence with at least 60% homology with SEQ ID NO: 1, an amino acid sequence with at least 75% homology with SEQ ID NO: 2, and an amino acid sequence with at least 90% homology with SEQ ID NO: 3; and said second enzyme is selected from the list comprising a fungal alpha-amylase (EC 3.2.1.1), a beta-amylase (E.C. 3.2.1.2), and a glucoamylase (E.C.3.2.1.3), to produce a soluble starch hydrolyzate, b) subjecting said soluble starch hydrolyzate to a conversion into fructose-rich corn syrup (HFCS). 18. Process for the production of a fermentation product, including the process the steps of: a) subjecting an aqueous suspension of granulated starch at a temperature below the initial gelatinization temperature of said granulated starch to the action of a first enzyme and a second enzyme, said first enzyme:

(i)

 it is an element of the 13 family of glycoside hydrolases;

(ii)

 It has alpha-1,4-glycosidic hydrolysis activity, and;

(iii) comprises a functional carbohydrate binding module (CBM) of the CBM family 20, said CBM has an amino acid sequence selected from the group consisting of an amino acid sequence with at least 60% homology with SEQ ID NO: 1, an amino acid sequence with at least 75% homology with SEQ ID NO: 2, and an amino acid sequence with at least 90% homology with SEQ ID NO: 3; and said second enzyme is selected from the list comprising a fungal alpha-amylase (EC 3.2.1.1), a beta-amylase (E.C. 3.2.1.2), and a glucoamylase (E.C.3.2.1.3), to produce a soluble starch hydrolyzate, b) subjecting said soluble starch hydrolyzate to fermentation in a fermentation product.

19.

Process according to claim 18 wherein the fermentation product is ethanol.

twenty.

Process according to any of claims 18 or 19, wherein the fermentation step is carried out simultaneously or separately / sequentially to the hydrolysis of the granulated starch.