JPH10512741A - Methods for increasing monosaccharide levels in starch saccharification and enzymes useful therefor - Google Patents

Abstract

(57) Abstract During the commercial production of dextrose from starch, the disaccharide maltulose is produced, which is hydrolyzed by the enzymes amyloglucosidase, pullulanase, or α-amylase. Can not be done. The present invention discloses an enzyme preparation for the hydrolysis of maltulose. Enzymatic hydrolysis of maltulose offers a new and additional method for improving monosaccharide levels in starch saccharification.

Description

DETAILED DESCRIPTION OF THE INVENTION          Methods for increasing monosaccharide levels in starch saccharification and          Enzymes useful for that                                Field of the invention   The present invention relates to the enzymatic degradation of starch. More specifically, the present invention relates to a liquefied Methods for obtaining increased monosaccharide levels from pun are provided.                                Background of the Invention   Native starch can contain two types of macromolecules composed of glucose units. And is known. One type of starch, amylose, is linear and exclusively Consists of glucose units linked by α-1,4 bonds. The second type, amylopectin Is highly branched and contains α-1,4 bonds in addition to α-1,4 bonds. The total content of α-1,6 bonds is generally less than 5%.   Sugar in the form of concentrated dextrose (glucose) syrup prepared from starch Is currently a two-step enzyme catalyzed process which involves: (1) converting starch into an α-amylase Involved in hydrolysis to dextrin with an average degree of polymerization of about 7-10 Liquefaction (or thinning) step, and (2) the resulting liquefied starch (de) Xistrin) is saccharified with amyloglucosidase, thereby increasing the high glucose content ( (92-96% of total solids weight). Commercially manufactured Many dextrose syrups are enzymatically known as iso-syrups. Dextrose / fructose mixture.   The two enzymes used, α-amylase and amyloglucosidase, are: They differ in two important aspects. First, α-amylase is a starch component. It is an endoenzyme that randomly attacks the inner bonds of offspring. On the other hand, amyloglucosider Ze is an exoenzyme that separates glucose units from the non-reducing end of dextrin molecules It is. Second, α-amylase almost exclusively attacks α-1,4 bonds. On the other hand, amyloglucosidase also has a ratio of α-1,6 bond to α-1,4 bond. It cuts at a significantly lower rate when compared.   The recommended name of amyloglucosidase is exo-1,4-α-D-glucosid ase (EC 3.2.1.3), strain name is α-1, 4 glucan glucohydrolar It is Ze. Amyloglucosidase is also called AG or glucoamylase It should be understood that these terms are equivalent when used hereinafter. It is.   The saccharification stage of commercial dextrose production is suboptimal in many respects. It has been recognized that. In particular, currently available amyloglucosidases are In the same manner as in the production reaction, the reverse reaction, such as dextrose to isomaltose, It also catalyzes conversion at a ratio depending on the source concentration. Formation of such by-products Demonstrated the saccharification of starch hydrolyzate to glucose by 33% d. s. (That is, Limited to a DX value of about 95% or less (in a syrup containing at least 33% dry solids) ing. The DX value is the weight percentage of dextrose on a carbohydrate dry solid basis. Is defined as   More efficient in liquefied starch in efforts to increase DX value To hydrolyze branched oligosaccharides (including α-1,6 linkages) The use of branching enzymes in combination with cosidases has been proposed. Europe Patent Application EP-A1-0063909 describes Bacillus acidopu pullulana produced by Bacillus called lllyticus Describes an enzyme-type branching enzyme. This method provides a DX increase of 0.4-0.6. Looks like it is bringing.   In another effort to further increase DX values, amyloglucosidase was used. For use with increased acid amylase levels (side activity present during AG repair) Has been proposed. This is described in European Patent EP B1-0140410. It is listed. In this method, 33% d. s. Results in a 0.5 increase in DX You.   Generally, high temperatures are used during the starch liquefaction process. These high temperatures In combination with the applied pH, the glue present at the reducing end of the dextrin molecule Stimulates isomerization to fructose in course units. The hydrolysis of dextrin is Free glucose, including glucose units (fructose) isomerized at the reducing end ー Disaccharides (glucose) called maltose and maltulose -Α-1,4-fructose). This disaccharide maltulose is an enzyme It is not hydrolyzed by AG, pullulanase and acid amylase, Remains in the dextrose syrup produced. During liquefaction Depending on the conditions used, the concentration of maltulose can be as high as 2% of the total sugar. Can get. The hydrolysis of the sugar maltulose is achieved by dextrose syrup and New and improved DX values in starch liquefaction for the production of syrups; Will provide additional methods. Maltulose is used in the fermentation process Not used, so maltulose can be fermented with glucose, fructose, etc. Conversion to a new sugar increases the fermentation yield of the starch saccharification process and reduces the primary A higher yield of product will be achieved.                                  Summary of the Invention   One object of the present invention is to convert non-fermentable sugars, especially maltulose, to glucose. Is useful in hydrolyzing to produce fermentable sugars such as To provide a useful enzyme.   A further object of the present invention is to reduce dextrose yield in the starch saccharification process. It is to provide a cost-effective way to increase it.   A still further object of the present invention is to provide the yield of primary metabolites of a fermentation step, such as ethanol. To provide a cost-effective way to increase the cost.   The present invention provides an enzyme preparation containing an enzyme capable of hydrolyzing maltulose. I do. In a preferred embodiment, the enzyme preparation is purified or qualitatively Includes an improved maturase enzyme. In a more preferred embodiment, the maturase The enzyme may be a fungal source, most preferably Aspergillus. Or derived from Trichoderma. specific In an embodiment of the composition of the invention, the maltulose enzyme is Aspergillus niger (A spergillus niger) and about 132 as determined by gel filtration. It has a molecular weight of kD.   In an embodiment of the method of the present invention, a method for enzymatic hydrolysis of maltulose is provided. A law is provided. The invention also increases the DX value of the sugar syrup produced To produce dextrose syrup with the enzyme preparation of the present invention. Provided is a method for saccharifying lop. The present invention further relates to a dextate comprising iso-syrup. Phosphorus and dextrose syrup, prepared from starch and maltose Are not included.                             BRIEF DESCRIPTION OF THE FIGURES Figure 1. Gel filtration chromatography of α-galactosidase on Sephacryl 5200HR. Matogram OD (OD280) vs elution time                                Description of the invention   The present invention provides for the use of enzymes present in liquid media obtained through microbial fermentation to When rutulose can be hydrolyzed to glucose and fructose It is based on the surprising discovery. Suitable yeast for the hydrolysis of maltulose The components are, for example, commercially available enzyme preparations, and preferably fungal cellulases and α- It can be derived from a galactosidase preparation. Maltulose hydrolysis activity, ie Has the ability to hydrolyse maltulose to, for example, glucose and fructose Such enzymes are hereinafter referred to as maltulase. Maltulase starts Standard proteins available to those skilled in the art from fermentation broth or enzyme preparations It can be purified using purification methods (eg, RK Scopes 1987, "P protein Purification, Principles and P ractice "Springer Verlag, New York, 2nd   edn).   As described herein, maltulase activity was unexpectedly found in Tricho commercially available fungal cells obtained from derma or Aspergillus species, respectively. Lulase and α-galactosidase preparations, but Lase can also be obtained from other fungi or other classes of microorganisms, and therefore It is justified that biologically derived maltulase generally falls within the scope of the present invention. It is. Techniques Obtained Through This Disclosure For Identification Of Maltulose Hydrolytic Activity As techniques are provided, one skilled in the art will appreciate any or many of the many commercially available enzyme preparations. Multiulase enzyme from the fermentation broth produced by keeping a number of microorganisms warm. Can be determined and purified.   Preferably, the maltulase is purified to homogeneity, whereby specific activity, molecular weight , Or partial or total polypeptide (s) having maltulase activity Determination of biochemical, physical and kinetic variables of enzymes, such as body amino acid sequences It is possible. The partial or complete amino acid sequence obtained is That enables molecular cloning of gene (s) encoding Used in the design of tides (Sambrook et al., 1989, "Mo rectangular Cloning: a laboratory manual " Cold Spring Harbor Laboratories, Col d Spring Harbor, New York, etc.). other Purified maltulase is used for gene cloning through expression cloning. Used to generate antibodies that allow Cloned multiler Ze gene (group) is, for example, Aspergillus, Trichoderma, Bacillus, Streptomyces, Saccharomyces, Or for the construction of overexpressing strains of industrial microorganisms such as Kluyveromyces (See, for example, Bennett and Lassure eds., 1991). "More Gene Manipulations in Fungi" Ac ademic Press Inc. , New York, etc.). These overproducers are the cost-effective and virtually endless of pure maltulase. Provide a source that is The resulting macromolecules, either through purification or overexpression strains Raturase preparations should be used to qualitatively enhance existing enzyme preparations Or in a form that is substantially pure, alone in a sugar solution containing maltulose Can be used for Substantially purified maltulase is preferred Will have additional enzymatic activity that is not important from a saccharification point of view.   A "quality-enhanced" maltulase preparation according to the invention is a maltulase preparation. From fermentation media produced by fermentation of naturally occurring microorganisms that produce A naturally-occurring preparation that is derived from a fermentation of a microorganism and is higher than would be found in nature. High maltulase concentration. Other qualitatively enhanced maltulase The preparation "qualitatively enhances" the maltulase compared to the contaminants removed As in the case of maltulase fermentation from the fermentation medium of a naturally or genetically engineered microorganism It can be prepared by purifying element. Similarly, the purified maturase enzyme is A naturally occurring enzyme mixture, such as pullulanase, glucoamylase or acid Enzymes are mixed with fermentation of naturally occurring organs (groups) to substances containing soluble amylase, etc. An object may be added at a higher concentration than is present in what is desired. Additional In some cases, a qualitatively enhanced maltulase preparation is used to Of genetically engineered microorganisms that have been treated recombinantly to amplify the expression of It may be derived by fermentation.   As mentioned above, the maltulase enzyme is available in many different enzyme preparations. Is believed to be seen in small quantities. However, commercially available enzyme preparations (ie, , A commercially available α-galactosidase). Thus, the amount of maltulase produced is generally the actual amount of starch processing. Inadequate for making improvements, or for starch substrates in the saccharification process In enzyme preparations that are not commonly applied (ie, commercially available cellulases). Especially preferred In another embodiment of the present invention, the mulch of the enzyme preparation in which it is used. Content increases its maltulose hydrolysis activity to commercially significant levels It has been in large quantities to make it happen. The added maltulase enzyme is It must be sufficient to substantially decompose the luchulose. Of course, the present invention The amount of maltulase enzyme added according to Depends on the amount of chulose. For example, 40% dry sugar with 2% maltulose In solution, maltulose is present in an amount of about 8 g / kg sugar. in this way If one unit is hydrolyzed at 1 μmole maltulose / min, then 6 units Per kg of syrup hydrolyzes maltulose in solution in 72 hours Is necessary for In addition, 432 units / kg can be used for mulching in solution in one hour. Necessary for hydrolyzing loin. Thus, preferably, the yeast of the present invention The maltulose hydrolysis activity of the sugar composition is about 1 unit / kg sugar d. s. In And more preferably 50-5000 units / kg sugar d. s. Between Also preferably 50-1000 units / kg sugar d. s. Between.   Matrix for hydrolysis of maltulose to monosaccharide glucose and fructose The use of luturase increases the DX value in the dextrose syrup of the product, The syrup is subsequently used as such or for the production of iso-syrup You. Enzymatic hydrolysis of maltulose reduces reduced maltulose levels, 0.5% or less of maltulose (percent of maltulose based on total sugar) Percent), or most preferably about 0.1% (ie, substantially maltulose). Dextrose syrup or iso-syrup) Bring. The hydrolysis of maltulose is preferably at a pH between about 3 and 7, More preferably at a pH between about 4 and 5, preferably between about 15 ° C and 70 ° C, and More preferably, it is performed at a temperature between about 20 ° C and 60 ° C.   The method for the hydrolysis of maltulose provided by the present invention is preferred. Alternatively, it is performed at the end of a conventional saccharification step. However, maltulose is a yeast Is not hydrolyzed by elemental AG, pullulanase, and acid amylase A further embodiment of the method of the invention is a combination with a method which is part of the state of the art. A method for utilizing maltulose is also provided. Thus, mulch Of pullulanase, glucoamylase or acid amylase in combination with Use will result in a further increase in DX values in the saccharification step. Marty The combined use of the enzyme, pullulanase and acid amylase Having a higher DX value than the combined use of Enables the preparation of sucrose syrup. In fact, the method of the present invention has up to two units. Improves DX level of dextrose syrup easily.   In a preferred embodiment, the maltulase enzyme is present in a liquefied starch solution. Used for hydrolysis of maltulose. Thus, for example, starch α- May be added to liquefied starch produced by jet liquefaction with amylase No. Alternatively, the maltulase enzyme is added in the saccharification step simultaneously with the glucoamylase. It may be. In still other variants, the maltulase enzyme is a liquefied Further increase DX value of saccharified starch after starch is treated with glucoamylase May be added to cause Finally, isomerized fructose / glucose Syrup is added to glucose and fructose to further increase It may be treated with maltulase in order to reduce the amount of the dulose mixture. these Each of the variants may benefit from the enzymes of the present invention through increased production of fermentable sugars. There will be. However, the choice of which variant to use in a given process The choice depends on the specific parameters under which the process will operate in the future. There will be. However, those skilled in the art will recognize any variation in a given starch treatment method. It is easy to see if the mold is optimal and the maltulose hydrolysis reaction Preferably during saccharification or after saccharification or during fermentation, it is This is because the process contains the highest maltulose concentration.   Maltulose hydrolysis means that dextrose syrup is carbon and / or The fermentation process used as a source of energy results in higher yields. One example of this is the fermentative production of ethanol from dextrose syrup. Maize in glucose syrup obtained through hydrolysis of starch to glucose Ethanol based on glucose syrup consumed due to the presence of luchulose The yield of knol will increase with the hydrolysis of maltulose. Maltulose Is hydrolyzed to fermentable sugars, biomass, (primary or secondary) metabolites , A similar increase in the yield of drugs (penicillins) or enzymes may also occur in other fermentation processes. Could happen. Thus, in a variant of the invention, dextrose Or iso-syrup is a microbial source of energy or primary metabolite Used in yeast.   The following examples are only intended to illustrate, but not limit, the invention. Should not be interpreted as                                  Example                                 Example 1               Analysis of commercial dextrose and iso-syrup   Dextrose and glucose / fructose made from starch Saw syrup contains maltulose. From commercially available dextrin mixtures The analysis was performed by processing different syrups produced by saccharification. sugar Amyloglucosidase (Amigase, Gi) at 60 ° C. and pH 4.2. (from st-brocades) for 48 hours. The resulting group The glucose syrup was subjected to a maltulose content by HPLC as described below. Was analyzed. The results of these analyzes are shown in Tables 1 and 2. Tables 1 and 2 As shown, the typical saccharification procedure applied to a commercial dextrin mixture is This resulted in a syrup containing luchulose.   Sugar concentration can be determined by high performance liquid chromatography. The preferred method is Use the following conditions: Column: Bio-Rad HPX 87C. Eluent: distilled water. Column temperature: 85 ° C. Flow rate ratio: 0.6 ml / min. Detection: RI (refractive index) detection.                                 Example 2                          Maltulose detection analysis                       (A) Preparation of maltulose   Maltulose is a disaccharide α-D-glucopyranosyl-1,4-α-fructov Lanose. This disaccharide is a disaccharide maltose (α-D-glue) as follows. Of the glucose residue at the reducing end of copyranosyl-1,4-α-glucopyranose) Can be prepared by alkaline isomerization: 2 g of aluminum oxide in 100 ml of 4 It was mixed with 0% (w / v) maltose solution. The pH is adjusted using sodium hydroxide H11.5. The reaction mixture was stored at 60 ° C. for 24 hours. Subsequently, the pH Was adjusted to pH 4.5, and 5 g of baker's yeast was stored in maltose and other alkaline solutions. Added to ferment fermentable sugars from the warming process (maltulose is Not fermented by yeast). Finally, the reaction mixture was filtered to get a clear solution, It is then concentrated under vacuum to remove ethanol (from fermentation), A highly dry solid solution of chulose was obtained.                  (B) Enzymatic hydrolysis of maltulose   Maltulose hydrolysis activity was investigated in a number of commercial enzyme preparations. enzyme The preparation was mixed with a 5% maltulose solution in distilled water. For each enzyme, 5 mg of enzyme was added to 5 ml of maltulose solution. The mixtures are listed in Table 2. Incubated under warm conditions. The reaction mixture was purified using HPLC as described in Example 1. And analyzed. Table 2 shows the results of these analyses.                                 Example 3        Effect of cellulase preparations on monosaccharide levels after saccharification by AG   To show the cellulase preparation relative to the monosaccharide level of the sugar solution after saccharification, Of cellulase was added to 10 ml of dextrose syrup. This dextro Sucrose syrup was prepared by saccharifying a 33% w / w dextrin solution with AG. Most Dextrose syrup water bath at 100 ° C for 10 minutes at large DX level The AG was inactivated by placing it inside. 3. Addition of cellulase and pH = 4. 5. After incubation for 1 hour at 50 ° C., 0.1 ml of the reaction mixture was distilled for 3 ml. By diluting with water and placing the diluted mixture in a boiling water bath The reaction was stopped. Finally, the sample was analyzed by HPLC as described in Example 1. Was analyzed as described. Table 3 shows the results. (DP is the degree of polymerization (Degree of polymerization) DP4 + means a degree of polymerization of 4 or higher. )   The results shown in Table 4 show that the addition of the cellulase preparation resulted in a monosaccharide level of 0.5- This indicates an increase of 0.6DX.                                 Example 4 Maltulose from Sumizyme AGS and Part of Residual Sugar Hydrolysis Activity Purification   α-galactosidase preparation (Sumizyme AGS, Shin Nih on, Japan) was partially purified using gel filtration chromatography. procedure Are described below. The collected fractions were searched for the presence of different activities. Interesting fractions were pooled for determination of specific activity. Chromatographic procedure: Column: 58x2.5cm Support material: Sephacryl S 200 HR Elution buffer: 50 mM acetate buffer, pH = 4.5, 0.02% azide Contains sodium Flow ratio: 2ml / min Detection: UV 280 nm Fraction collection: 2 minute fraction Sample: 4 ml of 30 mg / ml Sumizyme A in elution buffer GS (Lot 60902-02) Activity detection (fraction search): 1. α-galactosidase activity detection   100 μl of 1 mM paranitrophenyl in 50 mM acetate buffer pH 5.5 Le-α-D-galactose was incubated with 100 ml of the collected fractions. room temperature 100 μl of 0.0625 M Borax buffer pH after incubation for 3 minutes 9.7 was added to stop the reaction. Caused by para-nitrophenol The yellow color was measured as α-galactosidase activity. The results were judged visually. 2. Maltulose hydrolysis activity detection   The maltulose preparation was diluted four times with distilled water. Add 100 μl of this solution to 20 0 μl of the fraction having maltulose hydrolysis activity and 700 μl of distilled water Mixed. The mixture was kept at 33 ° C. for 3 hours. Next, to inactivate the enzyme The mixture was placed in a boiling water bath. Finally, the mixture is analyzed by HPLC for Bio -Analyzed using a Rad HPX 87C column. Fructose peak area Increase was a direct measure of activity. 3. Residual sugar hydrolysis activity   50 μl of the fraction having residual sugar hydrolysis activity was added to 50 μl beer (wet Fuel Ethanol Products from Pekin Energy, Pekin, Illi Noi) and incubated at 33 ° C. for 16 hours. Next, 900 μl of 0.006 N sulfuric acid was added and the reaction mixture was centrifuged in an Eppendorf centrifuge. The supernatant is HPL C analyzed on a BIO-Rad HPX 87H column. Residual sugar peak Area reduction was a direct measure of activity. Specific activity assay:   Specific activity was expressed as activity value / mg protein / minute reaction time. 1. Determination of protein content   Protein content was determined using the BCA method with bovine serum albumin as standard . 2. α-galactosidase activity   10 mM p-nitropheno in 50 mM sodium acetate buffer pH 5.5. A solution was prepared. This solution was diluted to 240-160-80-40 mM . 1 ml of these solutions was added to 0.80 mM p-nitro in 2 ml acetate buffer. Added to phenyl-α-D-galactopyranoside. Add 5 ml of 6 to this mixture 2 5 mM borax buffer pH 9.7 was added (stop reagent). These solutions The OD of the liquid was measured at 405 nm against water (standard curve). Incubation of enzyme: Add 1 ml of diluted enzyme solution instead of p-nitrophenol. Added. The mixture was incubated at 37 ° C. for 15 minutes. The reaction was performed with 5 ml of the Borax solution. The addition was stopped. OD was measured as described above. Definition of activity: 1 unit of α-galactosidase is 1 μmol of p-NPGa 1 / min is the amount of enzyme that hydrolyzes under standard conditions. 3. Maltulose hydrolysis activity   100 μl of the maltulose solution was mixed with 200 μl of the enzyme solution and 700 μl It was mixed with distilled water. The mixture was kept warm at 33 ° C. Samples are taken at different incubation times And analyzed by HPLC to determine the amount of hydrolyzed maltulose. 4. Residual sugar hydrolysis activity   100 μl of beer from wet mill fuel ethanol product was replaced with 200 μl of yeast The mixture was mixed with a hydrogen peroxide solution and kept at 33 ° C. 700 μl of 8 mM sulfuric acid in different incubations Added to samples taken at time. The sample was analyzed by HPLC for the amount of residual sugars hydrolyzed. Was analyzed to determine Definition of specific activity:   1. α-galactosidase: unit α-gal / mg protein   2. Maltulose hydrolysis activity: mg / m of hydrolyzed maltulose g protein / min   3. Residual sugar hydrolyzing activity: μg of hydrolyzed residual sugar / mg protein / min Determination of child size   A standard commercial protein mixture (Bio-Rad) was prepared using the following molecular weight Used as markers: thyroglobulin (MW = 670 kD), gamma globulin (MW = 158 kD), ovalbumin (MW = 44 KD), myoglobin (MW = 17 kD), vitamin B12 (MW = 13.5 kD). Gel filtration of markers and Subsequent comparisons with maltulase and residual sugar hydrolase show that The molecular weights of the other maltulase were about 132 kD and 120 kD. . result:   FIG. 1 is a chromatogram obtained by gel filtration of an α-galactosidase preparation. It is shown in FIG. Assay results for the presence of α-galactosidase activity in fractions, Maltulose hydrolysis activity and residual sugar hydrolysis activity are shown in Table 4 below. I have. This result was used to pool both minutes. α-galactosidase pool Was contained in fractions 8-10 (elution time = 55-60 minutes). Remnants-and Maltulose hydrolysis activity pool was fractions 12-14 (elution time = 63-68 min) A) was included. The specific activity of the pooled fractions and starting material was assayed. result Are shown in Table 5. Discussion / Conclusion   Tables 4 and 5 show that maltulose and residual sugar hydrolysis activity were Side activity in the enzyme preparation and is not due to α-galactosidase itself. It is not shown. In addition, the specific activity of both enzymes is It shows that it increases remarkably.                                 Example 5                     Heat inactivation of α-galactosidase   The solution of α-galactosidase was treated at 65 ° C. for 30 minutes. Next, the starting material and The heat treated solution was assayed for specific activity.   Table 6 shows the results of the experiment.   The results indicate that α-galactosidase activity and residue and maltulose addition This indicates that the hydrolytic activity comes from different enzymes.   The foregoing description and examples illustrate the invention, but do not limit the scope of the invention. It should be considered that the following claims define.

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Claims (1)

[Claims] 1. An enzyme preparation comprising an enzyme having maltulose hydrolysis ability, Enzyme preparation qualitatively improved for the maltulose hydrolyzing enzyme An enzyme preparation characterized in that: 2. The enzyme having the maltulose hydrolysis ability was obtained from a microorganism. An enzyme preparation according to claim 1, characterized in that: 3. 3. The enzyme preparation according to claim 2, wherein the microorganism is a fungus. Stuff. 4. The fungus is an Aspergillus strain or a Trichoderma strain. An enzyme preparation according to claim 3. 5. The enzyme preparation is derived from Aspergillus niger and is subjected to gel filtration. Includes a maltulase enzyme with a molecular weight of about 132 kD as measured An enzyme preparation according to claim 4, characterized in that: 6. Use of the enzyme preparation for maltulose hydrolysis. 7. Use of the enzyme preparation according to claim 1 for maltulose hydrolysis. 8. Using an enzyme preparation for the maltulose hydrolysis Maltulose hydrolysis method. 9. Dextrose, iso-syrup, and / or maltulose For producing glucose from a solution containing xistrin:     (A) containing dextrose, iso-syrup and / or dextrin Prepare a solution;     (B) Preparation of an enzyme containing an enzyme capable of hydrolyzing maltulose in the solution Wherein the enzyme preparation is capable of hydrolyzing the maltulose Qualitatively improved for enzymes having     (C) subjecting the solution from (b) to conditions suitable for maltulose hydrolysis and And heat for hours;   A method comprising the steps of: 10. Using the enzyme pullulanase, acid amylase, or a combination thereof 10. The method according to claim 9, further comprising: 11. Including the use of dextrose syrup produced according to claim 7 Fermentation process. 12. The fermentation process according to claim 11, for the fermentative production of ethanol. 13. Dextrose obtained from starch and substantially free of maltulose syrup. 14． Iso-syrup substantially free of maltulose obtained from starch .