EP0787202A1 - Verfahren zur erhoehung der monosaccharidniveau in der verzuckerung von staerke und enzyme fdafuer - Google Patents

Verfahren zur erhoehung der monosaccharidniveau in der verzuckerung von staerke und enzyme fdafuer

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Publication number
EP0787202A1
EP0787202A1 EP95941339A EP95941339A EP0787202A1 EP 0787202 A1 EP0787202 A1 EP 0787202A1 EP 95941339 A EP95941339 A EP 95941339A EP 95941339 A EP95941339 A EP 95941339A EP 0787202 A1 EP0787202 A1 EP 0787202A1
Authority
EP
European Patent Office
Prior art keywords
maltulose
enzyme
hydrolysis
enzyme preparation
starch
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP95941339A
Other languages
English (en)
French (fr)
Inventor
Bertus Noordam
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Danisco US Inc
Original Assignee
Genencor International Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genencor International Inc filed Critical Genencor International Inc
Priority to EP95941339A priority Critical patent/EP0787202A1/de
Publication of EP0787202A1 publication Critical patent/EP0787202A1/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06—Ethanol, i.e. non-beverage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/20—Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01022—Alpha-galactosidase (3.2.1.22)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention relates to the enzymatic degradation of starch. More specifically, the invention provides a method yielding increased monosaccharide levels from liquefied starch.
  • Native starch is known to contain two types of macromolecules composed of glucose units.
  • One type of starch amylose, is linear and consists of glucose units exclusively linked with ⁇ -1 ,4 bonds.
  • the second type, amylopectin is highly branched and contains ⁇ -1 ,6 bonds in addition to ⁇ -1 ,4 bonds.
  • the overall content of ⁇ -1 ,6 bonds is generally less than 5%.
  • Sugars prepared from starch in the form of concentrated dextrose (glucose) syrups are currently produced in a two stage enzyme catalysed process, involving: (1) a liquefaction step (or thinning) involving hydrolysis of starch with ⁇ -amylase into dextrins having an average degree of polymerization of about 7-10, and (2) saccharification of the resulting liquefied starch (dextrins) with amyloglucosidase, which results in a syrup having a high glucose content (92-96% by weight of total solids).
  • Much of the dextrose syrup produced commercially is enzymatically isomerized to a dextrose/fructose mixture, known as iso-syrup.
  • ⁇ -amylase is an endo-enzyme which attacks the internal linkages of starch molecules at random.
  • Amyloglucosidase is an exo-enzyme which splits glucose units from the non-reducing ends of dextrin molecules.
  • ⁇ -amylase almost exclusively attacks ⁇ -1 ,4 bonds whereas amyloglucosidase splits ⁇ -1 , 6 bonds as well, though at a considerably lower rate than ⁇ -1 ,4 bonds.
  • amyloglucosidase is exo-1 ,4- ⁇ -D-glucosidase (EC3.2.1.3) the systematic name is ⁇ -1 ,4 glucan glucohydrolase.
  • Amyloglucosidase is also called AG or glucoamylase and it should be understood that these terms, as used hereinafter, are synonymous.
  • the saccharification stage of commercial dextrose production has long been recognized to be sub-optimal in many respects.
  • the amyloglucosidases currently available catalyse glucose production reactions as well as reversion reactions, e.g. conversion of dextrose into isomaltose at a rate dependent on the glucose concentration.
  • DX value is defined as the percentage by weight of dextrose on a carbohydrate dry solids basis.
  • High temperatures are generally used during the starch liquefaction process. These high temperatures, in combination with the applied pH, stimulate isomerization of the glucose unit present at the reducing end of dextrin molecules into fructose. Hydrolysis of dextrins, including the isomerized glucose unit (fructose) at the reducing end, results in free glucose and a disaccharide, called maltulose (glucose- ⁇ -1 ,4-fructose). The disaccharide maltulose is not hydrolysed by the enzymes AG, pullulanase and acid amylase and, as a consequence, will remain in the dextrose syrup produced.
  • the concentration of maltulose can be as high as 2% of the total sugar.
  • Hydrolysis of the saccharide maltulose would provide a new and additional method for improvement of the DX value in the saccharification of starch for the production of dextrose syrups and iso-syrups.
  • maltulose is not utilized by microorganisms in fermentation processing, conversion of maltulose into a fermentable sugar, such as glucose and fructose, would increase the fermentable yield of starch saccharification processes and facilitate higher yields of primary metabolites such as ethanol.
  • the present invention provides for an enzyme preparation comprising an enzyme capable of the hydrolysis of maltulose.
  • the enzyme preparation comprises a purified or enriched maltulase enzyme.
  • the maltulase enzyme is derived from a fungal source, most preferably from Aspergillus or Trichoderma.
  • the maltulose enzyme is derived from Aspergillus niger and has a molecular weight of about 132 kD as measured by gel filtration.
  • a method for the enzymatic hydrolysis of maltulose is provided.
  • the invention also provides method for the saccharification of sugar syrups to produce dextrose syrups with the enzyme preparation of the invention so as to increase the DX value of the produced sugar syrup.
  • the invention further discloses dextrin and dextrose syrups, including iso- syrups, which are produced from starch and which are free of maltulose.
  • the present invention is based on the surprising discovery that maltulose can be hydrolysed into glucose and fructose by an enzyme present in broths obtained through microbial fermentation.
  • Suitable enzymes for the hydrolysis of maltulose may be derived from, for example, commercial enzyme preparations, and preferably fungal cellulase and ⁇ -galactosidase preparations.
  • the enzyme with maltulose hydrolysing activity i.e., having the ability to hydrolyze maltulose to, for example, glucose and frutose, is referred to as maltulase hereinafter.
  • the maltulase can be purified from fermentation broths or enzyme preparations using standard protein purification methods available to the person skilled in the art (see e.g. R.K.
  • maltulase activity was unexpectedly identified, as illustrated herein, in commercial fungal cellulase and ⁇ -galactosidase preparations obtained from Trichoderma or Aspergillus species, respectively, it is very probable that maltulases can be obtained from other fungi or even other classes of microorganisms and that therefore microbial maltulases in general fall within the scope of the present invention.
  • maltulase enzyme from any number of commercially available enzymes preparations or fermentation broths produced from incubations of many microorganisms.
  • the maltulase is purified to homogeneity, which allows determination of the biochemical, physical and kinetic parameters of the enzyme, such as specific activity, molecular weight, or the partial or full amino acid sequence(s) of the polypeptide(s) with maltulase activity.
  • the obtained partial or full amino acid sequences are used to design oligonucleotide probes which allow the molecular cloning of the gene(s) encoding the maltulase (see e.g. Sambrook et al. 1989, "Molecular Cloning: a laboratory manual” Cold Spring Harbour Laboratories, Cold Spring Harbour, New York).
  • the purified maltulase is used to raise antibodies which allow the cloning of the gene through expression cloning.
  • the cloned maltulase gene(s) is used to construct over-expressing strains of industrial microorganisms such as e.g.
  • the obtained maltulase preparation may be used to enrich an existing enzyme preparation or can be used in a substantially purified form by itself on a sugar solution which includes maltulose.
  • a substantially purified maltulase will preferably have insignificant additional enzyme activity in terms of saccharification.
  • An "enriched” maltulase preparation according to the present invention is a preparation which is derived from a fermentation broth produced by the fermentation of a naturally occurring microorganism which produces maltulase and which preparation includes a higher concentration of maltulase than would be found naturally from fermentation of the microorganism.
  • an enriched maltulase preparation can be prepared by purifying the maltulase enzyme from the fermentation broth of a natural or genetically engineered microorganism so as "enrich" the maltulase relative to the removed contaminants.
  • the purified maltulase enzyme can be added to a naturally occurring enzyme mixture containing, for example, pullulanase, glucoamylase or acid amylase, in a concentration greater than exists in the naturally occurring fermentation of the organism(s) from which the enzyme mixture is desired.
  • an enriched maltulase preparation may be derived from the fermentation of a genetically modified microorganism which has been subject to recombinant techniques so as to amplify expression of maltulase in a fermentation broth.
  • maltulase enzymes are found in many different enzyme preparations in small quantities.
  • the quantities of maltulase which are produced by microorganisms cultured under conditions to produce commercially available enzyme preparations are generally insufficient to achieve practical improvement in starch processing or are in enzyme preparations which are not generally applied to starch substrate at the saccharification stage (i.e., commercially available cellulase).
  • the maltulase content of an enzyme preparation in which it is employed is enriched to increase the maltulose hydrolyzing activity thereof to a commercially significant level.
  • the added maltulase enzyme should be sufficient to substantially hydrolyze the maltulose in solution.
  • the amount of added maltulase enzyme according to the invention will depend on the amount of maltulose in the starch or sugar solution. For example, in a 40% dry solids sugar solution containing 2% maltulose, maltulose will be present in a quantity of approximately 8 g/kg of sugar. Thus, where 1 unit equals the hydrolysis of I ⁇ mole of maltulose/minute, 6 U/kg of syrup will be needed to hydrolyze the maltulose in solution in 72 hours. Alternatively, 432 U/kg would be necessary to hydrolyze the maltulose in solution in 1 hour.
  • the maltulose hydrolyzing activity of the enzyme composition of the invention is greater than about 1 U/kg sugar d.s., more preferably between 50 and 5000 U/kg sugar d.s., and most preferably between 50 and 1000 U/kg sugar d.s.
  • the use of maltulase for the hydrolysis of maltulose into the monosaccharides glucose and fructose will increase the DX value in the production of dextrose syrups, which syrups can subsequently be used as such or for the production of iso-syrups.
  • the enzymatic hydrolysis of maltulose will result in dextrose syrups or iso-syrups with reduced maltulose levels, preferably less than 0.5 % maltulose (percentage maltulose of total sugar), or most preferably below the detection level of about 0.1%, (i.e. substantially free of maltulose).
  • the hydrolysis of maltulose should preferably take place between a pH of about 3 and 7, and more preferably between a pH of about 4 and 5; at temperatures preferably between about 15°C and 70 ⁇ C, and more preferably between about 20°C and 60°C.
  • the method for hydrolyzing maltalose provided by the present invention is preferably performed at the end of the conventional saccharification process.
  • maltulose cannot be hydrolysed by the enzymes AG, pullulanase and acid amylase
  • a further embodiment the method of the present invention provides for the use of maltulose in combination to the methods that are part of the state of the art.
  • the use of pullulanase, glucoamylase or acid amylase in combination with maltulase will effect a further increase in the DX value in saccharification processes.
  • the combined use of maltulase, pullulanase and acid amylase allows for the preparation of dextrose syrups with higher DX values than with the combined use of only pullulanase and acid amylase.
  • the method of the invention can easily improve the DX level of a dextrose syrup by up to 2 units.
  • the maltulase enzyme is utilized to hydrolyze maltulose in a liquefied starch solution.
  • the maltulase enzyme can be added to the liquefied starch produced by jet liquefaction of starch with ⁇ - amylase.
  • the maltulase enzyme can be added simultaneously with glucoamylase in the saccharification step.
  • the maltulase enzyme can be added after the liquefied starch has been treated with glucoamylase to further increase the DX value of the saccharified starch.
  • isomerized fructose/glucose syrups may be treated with the maltulase enzyme to further increase the concentration of glucose and fructose and reduce maltulose contaminant content.
  • the enzyme of the present invention through the increased production of fermentable sugars.
  • the choice of which variation to use in a given process will depend on the specific parameters under which the process at hand is operated. While those of skill in the art would be able to easily ascertain which variation is optimal with a given starch processing method, it is preferred that the maltulose hydrolyzing step occur during or after saccharification or during fermentation as these steps contain the highest concentration of maltulose.
  • Maltulose hydrolysis will result in a higher yield in fermentation processes in which dextrose syrups are used as a carbon and/or energy source.
  • An example of this is the fermentative production of ethanol from dextrose syrups. Because of the presence of maltulose in glucose syrups obtained through the hydrolysis of starch into glucose, the yield of ethanol based on glucose syrup consumed will increase upon hydrolysis of maltulose. Similar increases in yields of biomass, (primary or secondary) metabolites, drugs (penicillin) or enzymes will also occur in other fermentation processes were maltulose can be hydrolysed into a fermentable sugar.
  • a dextrose or iso-syrup is used as an energy source or source for primary metabolites in the fermentation of microorganisms.
  • Dextrose and glucose/fructose iso-syrups produced from starch contain maltulose.
  • the analysis was performed by subjecting different syrups which were produced by saccharification from commercially available dextrin mixtures. The saccharification was carried out at 60°C and pH 4.2 using amyloglucosidase (Amigase from Gist-brocades) for 48 hours. Samples from the resulting glucose syrups were analyzed for maltulose content by means of HPLC as described below. The results of these analyses are shown in Tables 1 and 2. As shown in Tables 1 and 2, common saccharification procedures applied to commercial dextrin mixtures results in syrups containing maltulose.
  • Saccharide concentrations can be detectedby high performance liquid chromtography.
  • a suitable method utilizes the following conditions: Column: Bio-Rad HPX 87C. Eluent: distilled water. Column temperature: 85°C. Flow rate: 0.6 ml/min. Detection: Rl (refractive index) detection. Table 1
  • Maltulose is the disaccharide ⁇ -D-glucopyranosyl-1 ,4- ⁇ -fructofuranose.
  • This disaccharide can be prepared by alkaline isomerization of the glucose residue at the reducing end of the disaccharide maltose ( ⁇ -D-glucopyranosyl 1 ,4-aglucopyranose) as follows: 2 g of aluminiumoxide are mixed with 100 ml of a 40% (w/v) maltose solution. The pH is adjusted to pH 11.5 using sodium hydroxide. The reaction mixture is kept at 60°C for 24 hours.
  • the pH is adjusted to pH 4.5 and 5 g of baker's yeast are added in order to ferment maltose and other fermentable sugars resulting from the alkaline incubation conditions (maltulose is not fermented by the yeast).
  • the reaction mixture is filtered to obtain a clear solution, which is concentrated under vacuum to remove the ethanol (resulting from the fermentation) and to obtain a high dry solids solution of maltulose.
  • Example 3 The effect of a cellulase preparation on the monosaccharide level after saccharification with AG
  • DP degree of polymerization
  • DP4+ means a polymerization degree of 4 or higher
  • Support material Sephacryl S 200 HR.
  • Sample 4 ml of 30 mg/ l Sumizyme AGS (lot 60902-02) in elution buffer.
  • ⁇ -Galactosidase activity detection 100 ⁇ l 1 mM paranitrophenyl- ⁇ -D-galactose in 50 mM acetate buffer pH 5.5 was incubated with 100 ml of collected fraction. After a 3 minute incubation at room temperature 100 ⁇ l of 0.0625 M borax buffer pH 9.7 was added to stop the reaction. The yellow colour, resulting from paranitrophenol, was a measure for ⁇ - galactosidase activity. The result was judged visually. 2. Maltulose hydrolysing activity detection.
  • the maltulose preparation was diluted 4 times with distilled water. 100 ⁇ J of this solution were mixed with 200 ⁇ l of a fraction having maltulose hydrolyzing activity and 700 ⁇ l of distilled water. This mixture was incubated for 3 hours at 33 C C. Next, the mixtures were placed in a boiling water bath in order to inactivate the enzyme.
  • the specific activity is expressed as an activity value per mg of protein per minute of reaction time. 1. Determination of the protein content.
  • the protein content is determined using the BCA method with bovine serum albumin as standard. 2.
  • a solution is made of 10 mM p-nitrophenol in 50 mM sodium acetate buffer pH 5.5. This solution is diluted to 240-160-80-40 mM. 1 ml of these solutions is added to 2 ml of 0.80 mM p-nitrophenyl- ⁇ -D-galactopyranoside in acetate buffer. To this mixture 5 ml 625 mM borax buffer pH 9.7 is added (stop reagent). The OD of these solutions is measured at 405 nm against water (standard curve).
  • Enzyme incubation 1 ml of diluted enzyme solution in stead of p-nitrophenol. The mixture is incubated for 15 minutes at 37°C. The reaction is stopped by adding 5 ml borax solution. The OD is measured as mentioned before.
  • Activity definition one Unit of ⁇ -galactosidase is the amount of enzyme which hydrolyses 1 ⁇ mol of p-NPGal/minute under the standard conditions. 3. The maltulose hydrolysing activity.
  • maltulose solution 100 ⁇ l of maltulose solution is mixed with 200 ⁇ l of enzyme solution and 700 ⁇ l of distilled water. The mixture is incubated at 33 ⁇ C. Samples were taken at different incubation times and analyzed by HPLC in order to determine the amount of maltulose hydrolysed.
  • ⁇ -galactosidase units ⁇ -gal per mg protein.
  • Tables 4 and 5 demonstrate that the maltulose and residual sugar hydrolysing activity are side activities in the ⁇ -galactosidase preparation and are not due to the ⁇ -galactosidase itself. In addition, it appears that the specific activity of both enzymes can be significantly increased by a single purification step.
  • a solution of ⁇ -galactosidase was heat treated for 30 minutes at 65°C. Next, the starting material and the heat treated solution were assayed for specific activity.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
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  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • General Health & Medical Sciences (AREA)
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  • Biotechnology (AREA)
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  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
EP95941339A 1994-10-27 1995-10-26 Verfahren zur erhoehung der monosaccharidniveau in der verzuckerung von staerke und enzyme fdafuer Withdrawn EP0787202A1 (de)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP95941339A EP0787202A1 (de) 1994-10-27 1995-10-26 Verfahren zur erhoehung der monosaccharidniveau in der verzuckerung von staerke und enzyme fdafuer

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
EP94203124 1994-10-27
EP94203124 1994-10-27
PCT/US1995/013879 WO1996013602A1 (en) 1994-10-27 1995-10-26 A method for increasing monosaccharide levels in the saccharification of starch and enzymes useful therefor
EP95941339A EP0787202A1 (de) 1994-10-27 1995-10-26 Verfahren zur erhoehung der monosaccharidniveau in der verzuckerung von staerke und enzyme fdafuer

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EP0787202A1 true EP0787202A1 (de) 1997-08-06

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EP95941339A Withdrawn EP0787202A1 (de) 1994-10-27 1995-10-26 Verfahren zur erhoehung der monosaccharidniveau in der verzuckerung von staerke und enzyme fdafuer

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EP (1) EP0787202A1 (de)
JP (1) JPH10512741A (de)
KR (1) KR970707294A (de)
CN (1) CN1166860A (de)
AU (1) AU4278896A (de)
CA (1) CA2203812A1 (de)
FI (1) FI971782A (de)
WO (1) WO1996013602A1 (de)

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CN108474016A (zh) * 2016-01-05 2018-08-31 卡吉尔公司 用于发酵糖的方法

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Publication number Priority date Publication date Assignee Title
DE3475209D1 (en) * 1983-09-11 1988-12-22 Gist Brocades Nv Novel enzyme product and its use in the saccharification of starch
US4717662A (en) * 1985-01-31 1988-01-05 Miles Laboratories, Inc. Thermal stabilization of alpha-amylase
ES2052565T3 (es) * 1986-07-09 1994-07-16 Novo Nordisk As Un procedimiento para licuar una suspension de almidon o granos amilaceos.
US5231017A (en) * 1991-05-17 1993-07-27 Solvay Enzymes, Inc. Process for producing ethanol

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Title
See references of WO9613602A1 *

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FI971782A0 (fi) 1997-04-25
KR970707294A (ko) 1997-12-01
AU4278896A (en) 1996-05-23
FI971782A (fi) 1997-04-25
CA2203812A1 (en) 1996-05-09
JPH10512741A (ja) 1998-12-08
WO1996013602A1 (en) 1996-05-09
CN1166860A (zh) 1997-12-03

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