WO2002038786A1 - Ethanol process - Google Patents
Ethanol process Download PDFInfo
- Publication number
- WO2002038786A1 WO2002038786A1 PCT/DK2001/000709 DK0100709W WO0238786A1 WO 2002038786 A1 WO2002038786 A1 WO 2002038786A1 DK 0100709 W DK0100709 W DK 0100709W WO 0238786 A1 WO0238786 A1 WO 0238786A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- alpha
- amylase
- whole grain
- xylanase
- cellulase
- Prior art date
Links
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 88
- 238000000034 method Methods 0.000 title claims description 77
- 108090000637 alpha-Amylases Proteins 0.000 claims abstract description 72
- 102000004139 alpha-Amylases Human genes 0.000 claims abstract description 71
- 229940024171 alpha-amylase Drugs 0.000 claims abstract description 60
- 102000004190 Enzymes Human genes 0.000 claims abstract description 53
- 108090000790 Enzymes Proteins 0.000 claims abstract description 53
- 229940088598 enzyme Drugs 0.000 claims abstract description 53
- 108010059892 Cellulase Proteins 0.000 claims abstract description 34
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims abstract description 31
- 108010059820 Polygalacturonase Proteins 0.000 claims abstract description 28
- 229940106157 cellulase Drugs 0.000 claims abstract description 28
- 108010093305 exopolygalacturonase Proteins 0.000 claims abstract description 28
- 108010083879 xyloglucan endo(1-4)-beta-D-glucanase Proteins 0.000 claims abstract description 26
- 239000006188 syrup Substances 0.000 claims abstract description 20
- 235000020357 syrup Nutrition 0.000 claims abstract description 20
- 239000000203 mixture Substances 0.000 claims abstract description 19
- 230000000694 effects Effects 0.000 claims description 47
- 235000020985 whole grains Nutrition 0.000 claims description 31
- 239000002002 slurry Substances 0.000 claims description 25
- 238000000855 fermentation Methods 0.000 claims description 21
- 230000004151 fermentation Effects 0.000 claims description 21
- 102100022624 Glucoamylase Human genes 0.000 claims description 15
- 108010073178 Glucan 1,4-alpha-Glucosidase Proteins 0.000 claims description 12
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 11
- 241000228212 Aspergillus Species 0.000 claims description 9
- 240000008042 Zea mays Species 0.000 claims description 8
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 8
- 238000004821 distillation Methods 0.000 claims description 8
- 244000005700 microbiome Species 0.000 claims description 8
- 239000000463 material Substances 0.000 claims description 7
- 102000004169 proteins and genes Human genes 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 6
- 239000008186 active pharmaceutical agent Substances 0.000 claims description 6
- 229910052799 carbon Inorganic materials 0.000 claims description 6
- 235000005822 corn Nutrition 0.000 claims description 6
- 241000228215 Aspergillus aculeatus Species 0.000 claims description 5
- 230000007062 hydrolysis Effects 0.000 claims description 5
- 238000006460 hydrolysis reaction Methods 0.000 claims description 5
- 238000003801 milling Methods 0.000 claims description 5
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 4
- 241001480714 Humicola insolens Species 0.000 claims description 4
- 240000006394 Sorghum bicolor Species 0.000 claims description 4
- 235000011684 Sorghum saccharatum Nutrition 0.000 claims description 4
- 235000021307 Triticum Nutrition 0.000 claims description 3
- -1 cellulanase Proteins 0.000 claims description 3
- 239000002031 ethanolic fraction Substances 0.000 claims description 3
- 240000005979 Hordeum vulgare Species 0.000 claims description 2
- 235000007340 Hordeum vulgare Nutrition 0.000 claims description 2
- 240000003183 Manihot esculenta Species 0.000 claims description 2
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 claims description 2
- 241000235070 Saccharomyces Species 0.000 claims description 2
- 244000061456 Solanum tuberosum Species 0.000 claims description 2
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 2
- 235000009430 Thespesia populnea Nutrition 0.000 claims description 2
- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 235000009973 maize Nutrition 0.000 claims description 2
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims 1
- 244000098338 Triticum aestivum Species 0.000 claims 1
- 239000000727 fraction Substances 0.000 claims 1
- 238000004519 manufacturing process Methods 0.000 abstract description 14
- 239000000758 substrate Substances 0.000 description 21
- 229920002472 Starch Polymers 0.000 description 18
- 239000000243 solution Substances 0.000 description 17
- 235000019698 starch Nutrition 0.000 description 17
- 239000008107 starch Substances 0.000 description 17
- 108091005804 Peptidases Proteins 0.000 description 14
- 239000004365 Protease Substances 0.000 description 14
- 230000002538 fungal effect Effects 0.000 description 13
- 102000035195 Peptidases Human genes 0.000 description 11
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 11
- 239000000047 product Substances 0.000 description 11
- 238000002835 absorbance Methods 0.000 description 9
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- 241000228245 Aspergillus niger Species 0.000 description 6
- 102100024295 Maltase-glucoamylase Human genes 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 108010028144 alpha-Glucosidases Proteins 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Substances CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 101710146708 Acid alpha-amylase Proteins 0.000 description 4
- 239000004382 Amylase Substances 0.000 description 4
- 108010084185 Cellulases Proteins 0.000 description 4
- 102000005575 Cellulases Human genes 0.000 description 4
- 229920001353 Dextrin Polymers 0.000 description 4
- 239000004375 Dextrin Substances 0.000 description 4
- 108050008938 Glucoamylases Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 230000002378 acidificating effect Effects 0.000 description 4
- 235000013339 cereals Nutrition 0.000 description 4
- 235000019425 dextrin Nutrition 0.000 description 4
- 238000009837 dry grinding Methods 0.000 description 4
- 239000000446 fuel Substances 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 238000001238 wet grinding Methods 0.000 description 4
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical group [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 3
- 240000006439 Aspergillus oryzae Species 0.000 description 3
- 239000006171 Britton–Robinson buffer Substances 0.000 description 3
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- 241000233866 Fungi Species 0.000 description 3
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 3
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- 150000008163 sugars Chemical class 0.000 description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 2
- UHPMCKVQTMMPCG-UHFFFAOYSA-N 5,8-dihydroxy-2-methoxy-6-methyl-7-(2-oxopropyl)naphthalene-1,4-dione Chemical compound CC1=C(CC(C)=O)C(O)=C2C(=O)C(OC)=CC(=O)C2=C1O UHPMCKVQTMMPCG-UHFFFAOYSA-N 0.000 description 2
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- 241000894006 Bacteria Species 0.000 description 2
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 241000193403 Clostridium Species 0.000 description 2
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- 241000223218 Fusarium Species 0.000 description 2
- 241000193385 Geobacillus stearothermophilus Species 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 229920002488 Hemicellulose Polymers 0.000 description 2
- 101001091385 Homo sapiens Kallikrein-6 Proteins 0.000 description 2
- 102100034866 Kallikrein-6 Human genes 0.000 description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 2
- 241000228143 Penicillium Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 2
- 241000223259 Trichoderma Species 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
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- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- 239000001632 sodium acetate Substances 0.000 description 2
- 235000017281 sodium acetate Nutrition 0.000 description 2
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- 241000894007 species Species 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
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- 108010011619 6-Phytase Proteins 0.000 description 1
- 235000019890 Amylum Nutrition 0.000 description 1
- 101000961203 Aspergillus awamori Glucoamylase Proteins 0.000 description 1
- 101000756530 Aspergillus niger Endo-1,4-beta-xylanase B Proteins 0.000 description 1
- 101000757144 Aspergillus niger Glucoamylase Proteins 0.000 description 1
- 241000228251 Aspergillus phoenicis Species 0.000 description 1
- 241000228232 Aspergillus tubingensis Species 0.000 description 1
- 241000193744 Bacillus amyloliquefaciens Species 0.000 description 1
- 241000194110 Bacillus sp. (in: Bacteria) Species 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 241001509321 Clostridium thermoamylolyticum Species 0.000 description 1
- 241000222356 Coriolus Species 0.000 description 1
- 101100317179 Dictyostelium discoideum vps26 gene Proteins 0.000 description 1
- 101100407639 Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) prtB gene Proteins 0.000 description 1
- 241001246273 Endothia Species 0.000 description 1
- 241000588698 Erwinia Species 0.000 description 1
- 241000223198 Humicola Species 0.000 description 1
- OAKJQQAXSVQMHS-UHFFFAOYSA-N Hydrazine Chemical compound NN OAKJQQAXSVQMHS-UHFFFAOYSA-N 0.000 description 1
- 241000222342 Irpex Species 0.000 description 1
- 241000157919 Jonesia Species 0.000 description 1
- 241000228423 Malbranchea Species 0.000 description 1
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- 239000005913 Maltodextrin Substances 0.000 description 1
- 241000235395 Mucor Species 0.000 description 1
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- 125000003275 alpha amino acid group Chemical group 0.000 description 1
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- 230000003625 amylolytic effect Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
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- MCPLVIGCWWTHFH-UHFFFAOYSA-M disodium;4-[4-[[4-(4-sulfoanilino)phenyl]-[4-(4-sulfonatophenyl)azaniumylidenecyclohexa-2,5-dien-1-ylidene]methyl]anilino]benzenesulfonate Chemical group [Na+].[Na+].C1=CC(S(=O)(=O)O)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[NH+]C=2C=CC(=CC=2)S([O-])(=O)=O)C=2C=CC(NC=3C=CC(=CC=3)S([O-])(=O)=O)=CC=2)C=C1 MCPLVIGCWWTHFH-UHFFFAOYSA-M 0.000 description 1
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- 238000011084 recovery Methods 0.000 description 1
- 235000020071 rectified spirit Nutrition 0.000 description 1
- KUIXZSYWBHSYCN-UHFFFAOYSA-L remazol brilliant blue r Chemical compound [Na+].[Na+].C1=C(S([O-])(=O)=O)C(N)=C2C(=O)C3=CC=CC=C3C(=O)C2=C1NC1=CC=CC(S(=O)(=O)CCOS([O-])(=O)=O)=C1 KUIXZSYWBHSYCN-UHFFFAOYSA-L 0.000 description 1
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- 230000001131 transforming effect Effects 0.000 description 1
- IEDVJHCEMCRBQM-UHFFFAOYSA-N trimethoprim Chemical compound COC1=C(OC)C(OC)=CC(CC=2C(=NC(N)=NC=2)N)=C1 IEDVJHCEMCRBQM-UHFFFAOYSA-N 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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
- 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)
-
- 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)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
-
- 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)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
-
- 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)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
-
- 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 an improved process of producing ethanol; the use of enzyme activity for thinning the liquefied whole grain mash and the Thin Stillage and/or condensate thereof (evaporated Thin Stillage) and/or syrup thereof; a composition comprising alpha-amylase, xylanase, xyloglucanase, cellulase, pectinase, or a mixture thereof, suitably reducing the viscosity of mash and of Thin Stillage and/or condensate thereof and/or syrup thereof.
- Ethanol has widespread application as an industrial chemical, gasoline additive or straight liquid fuel.
- ethanol dramatically reduces air emissions while improving engine performance.
- ethanol reduces national dependence on finite and largely foreign fossil fuel sources while decreasing the net accumulation of carbon dioxide in the atmosphere.
- Fermentation processes are used for the production of ethanol.
- Fig. 1 schematically shows an ethanol production process of the invention.
- the invention relates to an improved process of producing ethanol as will be described below.
- Ethanol production In ethanol production processes the starting raw material is in a preferred embodiment of the invention whole grain. However, the side-products from starch conversion processes may also be used. A wide variety of starch containing whole grain crops may be used as raw material including: corn (maize), milo, potato, cassava, sorghum, wheat, and barley.
- the main process steps can be separated into the following main process stages: - Milling,
- the individual process steps of alcohol production may be performed batch wise or as a continuous flow.
- processes where one or more process step(s) is(are) performed batch wise or one or more process step(s) is(are) performed as a continuous flow are equally contemplated.
- contemplated are also processes where the fermentation step is performed as a continuous flow.
- the cascade process is an example of a process where one or more process step(s) is(are) performed as a continuous flow and as such contemplated for the invention. Further information on the cascade process and other ethanol processes can be found in, e.g., "The Alcohol Textbook” Eds. T.P. Lyons, D.R. Kesall and J.E. Murtagh. Nottingham University Press 1995.
- Milling The whole grain is milled in order to open up the structure and allowing for further processing.
- Two processes can be used: wet and dry milling.
- Preferred for ethanol production at least according to the present invention, is dry milling where the whole kernel is milled and used in the remaining part of the process.
- Wet milling gives a good separation of germ and meal (starch granules and protein) and is with a few exceptions applied at locations where there is a parallel production of syrups. Both dry and wet milling is well known in the art of ethanol production. Both dry and wet milling is contemplated for the present invention.
- milled gelatinized starch whole grain raw material is broken down (hydrolyzed) into maltodextrins (dextrins) mostly of a DP higher than 4.
- the hydrolysis may be carried out by acid treatment or enzymatically by alpha-amylase. Acid hydrolysis is used on a limited basis.
- the raw material is in one embodiment milled whole grain or a side stream from starch processing.
- enzymatic liquefaction is carried out as a three-step hot slurry process.
- the slurry is heated to between 60-95°C, preferably 80-85°C, and the en- zyme(s) is (are) added to initiate liquefaction (thinning).
- the slurry is jet-cooked at a temperature between 95-140°C, preferably 105-125°C to complete gelanitization of the slurry.
- the slurry is cooled to 60-95°C and more enzyme(s) is(are) added to finalize hydrolysis.
- the liquefaction process is carried out at pH 4.5-6.5, in particular at a pH between 5 and 6. Milled and liquefied whole grain is also known as mash. Saccharification
- the maltodex- trin from the liquefaction must be further hydrolyzed.
- the hydrolysis is typically done enzy- matically in the presence of a glucoamylase (AMG).
- AMG glucoamylase
- An alpha-glucosidase and/or an acid alpha-amylase may also be present.
- a full saccharification step may last up to 72 hours, however, it is common only to do a pre-saccharification of typically 40-90 minutes and then complete saccharification during fermentation (SSF). Saccharification may be carried out at temperatures from 30-65°C, in particular around 60°C, and at a pH in the range between 4-5, especially around pH 4.5.
- Yeast typically from Saccharomyces spp., in particular Saccharomyces cerevisiae or bakers yeast, is added to the mash and the fermentation is ongoing for 24-96 hours, such as 35-60 hours.
- the temperature is between 26-34°C, in particular at about 32°C, and at pH 3-6, in particular around pH 4-5.
- the most widely used process is a simultaneous saccharification and fermentation (SSF) process where there is no holding stage for the saccharification, meaning that yeast and enzyme is added together.
- SSF simultaneous saccharification and fermentation
- the mash is distilled to extract the ethanol.
- the ethanol obtained according to the process of the invention may be used as, e.g., fuel ethanol; drinking ethanol, i.e., potable neutral spirits; or industrial ethanol.
- the aqueous by-product (Whole Stillage) from the distillation process is separated into two fractions, for instance by centrifugation: 1) Wet Grain (solid phase), and 2) Thin Stillage (Supernatant).
- the Wet Grain fraction is dried, typically in a drum dryer.
- the dried product is referred to as "Distillers Dried Grain", and can be used as animal feed.
- the Thin Stillage fraction may be evaporated providing two fractions: - condensate fraction of 4-6% DS (mainly of starch, proteins, and cell wall components), and - Syrup fraction, mainly consisting of limit dextrins and non fermentable sugars, which may be introduced into a dryer together with the Wet Grain (from the Whole Stillage separation step) to provide a product referred to as "Distillers Dried Grain", which can be used as animal feed.
- “Whole Stillage” is the term used in the art for the side-product (main product is ethanol) coming from the distillation of fermented mash (see Fig. 1).
- Thin Stillage is the term used in the art for the supernatant of the centrifugation of the Whole Stillage.
- the Thin Stillage contains 4-6% DS (mainly starch and proteins) and has a temperature of about 60-90°C.
- Thin Stillage is viscous and difficult to handle.
- Thin Stillage is normally kept in a holding tank for up to a few hours before recycling to the slurry tank (see Fig. 1).
- the invention relates to a process of producing ethanol, wherein liquefied whole grain mash is thinned by treatment with an efficient amount of enzyme activity se- lected from the group consisting of alpha-amylase, xylanase, xyloglucanase, cellulase, pectinase, and mixtures thereof.
- the invention relates to a process of producing ethanol, wherein the Thin Stillage and/or condensate and/or syrup of evaporated Thin Stillage is thinned by treatment with an efficient amount of enzyme activity selected from the group consisting of alpha- amylase, xylanase, xyloglucanase, cellulase, pectinase, and mixtures thereof.
- the treated or untreated Thin Stillage and/or condensate and/or syrup of evaporated Thin Stillage is recycled to the liquefaction step, in particular to the milled whole grain slurry.
- Fig. 1 A specific ethanol production process (system) is shown in Fig. 1.
- the invention relates to a process for the production of ethanol which comprise the following steps: a) milling whole grain; b) liquefying gelatinised whole grain in the presence of an alpha-amylase; c) saccharifying the liquefied material in the presence of a glucoamylase; d) fermentation with a micro-organism; e) distillation of the fermented and saccharified material, providing two fractions: 1) alcohol fraction, and 2) Whole Stillage fraction;
- the Wet Grain fraction is dried to provide a protein containing product referred to as "Distillers Dried Grain", which is used as an animal feed product;
- the Thin Stillage may be evaporated providing two fractions: 1) Condensate fraction, and 2) Syrup fraction (mainly consisting of limit dextrin and non-fermentable sugars), which is introduced into a (drum) dryer together with the Wet Grain fraction (from the Whole Stillage separation step) to provide a product referred to as "Distillers Dried Grain", which is used as animal feed.
- the liquefaction step comprising the following sub-steps: b1) the hot slurry is heated to between 60-95°C, preferably 80-85°C, and at least an alpha- amylase is added; b2) the slurry is jet-cooked at a temperature between 95-140°C, preferably 105-125°C to complete gelanitization of the slurry; b3) the slurry is cooled to 60-95°C and more alpha-amylase is added to finalize hydrolysis.
- the DS% (dry solid percentage) in the slurry tank is in the range from 1-60%, in particular 10-50%, such as 20-40%), such as 25-35%.
- the liquefaction process is in an embodiment carried out at pH 4.5-6.5, in particular at a pH between 5 and 6.
- the thinning of the mash is preferably carried out or initiated as early in the process as possible.
- the thinning enzyme(s) is(are) added to the liquefied mash during or after the secondary liquefaction step, Alternatively the thinning en- zyme(s) is(are) added to the mash at the mash cooling step.
- the thinning enzyme(s) are added to the mash during pre-saccharification, saccharification, fermentation, or SSF.
- Steps (c) and (d) i.e., saccharification and fermentation
- Steps (c) and (d) may be carried out either simul- taneously or separately/sequential.
- an optional ethanol recovery step may be added.
- the fermented and according to the invention thinned mash is distilled to provide an ethanol fraction as described above.
- the ethanol fraction may optionally be refined further.
- the Thin Stillage from step (f2) and/or condensate and/or syrup of Thin Stillage from step (g2) may be recycled to the liquefaction step, in particular to the slurry containing the milled whole grain to be jet cooked.
- the Thin Stillage from step (f2) or the condensate from step (g2) is subjected to an effective amount of enzyme activity selected from the group consisting of alpha-amylase, xylanase, xyloglucanase, cellulase, pectinase, and mixtures thereof.
- enzyme activity selected from the group consisting of alpha-amylase, xylanase, xyloglucanase, cellulase, pectinase, and mixtures thereof.
- the enzyme treatment is preferably performed in the holding tank.
- the term "thinning enzyme(s)" used above means that the liquefied mash and/or the Thin Stillage and/or condensate and/or syrup of Thin Stillage is subjected to an effective amount of enzyme selected from the group of alpha-amylase, xylanase, xyloglucanase, cellulase, pectinase activity, or mixtures thereof.
- the treated thin stillage and/or condensate and/or syrup is recycled to the liquefaction step b), in particular to the (gelatinized) milled whole grain slurry to be jet cooked.
- Thin Stillage is not recycled, but the condensate stream and/or the syrup of evaporated Thin Stillage is recycled to the liquefaction step, in particular to the slurry containing the milled whole grain to be jet cooked.
- the Thin Stillage and/or the condensate and/or the syrup of evaporated Thin Stillage is subjected to an effective amount of an enzyme activity selected from the group consisting of alpha-amylase, xylanase, xyloglucanase, cellulase, pectinase, and mixtures thereof.
- an enzyme activity selected from the group consisting of alpha-amylase, xylanase, xyloglucanase, cellulase, pectinase, and mixtures thereof.
- Mixtures contemplated include the following combination: alpha-amylase and xy- lanase activity; alpha-amylase and cellulase activity; alpha-amylase and pectinase activity; xylanase and cellulase activity; xylanase and pectinase activity; cellulase and pectinase activity; alpha-amylase, xylanase and cellulase activity; alpha-amylase, xylanase and pectinase activity; xylanase, cellulase and pectinase activity; alpha-amylase, cellulase and pectinase activity.
- additional thinning enzymes in particular hemicellulose degrad- ing enzymes, such as xyloglucanases are added to the whole grain slurry.
- the viscosity of the whole grain slurry is, in part, a result of the hemicellulose contribution to the overall viscosity.
- xyloglucanases the structure of dry milled corn is altered, more closely resembling a corn that has been extremely and efficiently fine milled to a smaller particle size.
- Various en- zymatic systems have been tested showing that it is possible to process corn at a higher dry solid content, >40% DS vs. current 35-38% DS, while maintaining an acceptable viscosity for further processing.
- Liquefaction of the gelatinized milled whole grain slurry may be performed in the pres- ence of an alpha-amylase derived from a micro-organism or a plant.
- Preferred alpha-amylases are of fungal or bacterial origin.
- Bacillus alpha-amylases (often referred to as "Termamyl-like alpha-amylases"), variant and hybrids thereof, are specifically contemplated according to the invention.
- Well-known Termamyl-like alpha-amylases include alpha-amylase derived from a strain of B. licheniformis (commercially available from Novozymes A/S as TermamylTM), B. amyloliquefaciens, and B.
- Termamyl-like alpha- amylases include alpha-amylase derived from a strain of the Bacillus sp. NCIB 12289, NCIB 12512, NCIB 12513 or DSM 9375, all of which are described in detail in WO 95/26397, and the alpha-amylase described by Tsukamoto et al., Biochemical and Biophysical Research Communications, 151 (1988), pp. 25-31.
- a Termamyl- like alpha-amylase is an alpha-amylase as defined in WO 99/19467 on page 3, line 18 to page 6, line 27.
- Contemplated variants and hybrids are described in WO 96/23874, WO 97/41213, and WO 99/19467.
- Contemplated alpha-amylase derived from a strain of Aspergillus includes Aspergillus oryzae and Aspergillus niger alpha-amylases.
- Specifically contemplated variants include: Bacillus stearothermophilus alpha-amylase variants disclosed in WO 99/19467.
- alpha-amylase products and products containing alpha-amylases include TERMAMYLTM SC, FUNGAMYLTM, LIQUOZYMETM SC and SANTM SUPER, (from Novozymes A/S) and DEX-LOTM, SPEZYMETM AA, and SPEZYMETM DELTA AA (from Genencor Int.)
- contemplated alpha-amylases include the KSM-K36 alpha-amylase disclosed in
- Treatment of the Thin Stillage and/or condensate and/or syrup of evaporated Thin Stillage according to the process of the invention may be carried out in the presence of an effective amount of any of the above-mentioned alpha-amylases.
- Treatment of the Thin Stillage and/or condensate and/or syrup of evaporated Thin Stillage may be carried out in the presence of an effective amount of any of the following xylanases.
- Xylanase activity may be derived from any suitable organism, including fungal and bacterial organisms, such as Aspergillus, Disporo- trichum, Penicillium, Neurospora, Fusarium and Trichoderma.
- xylanases examples include xylanases derived from H. insolens (WO 92/17573; Aspergillus tubigensis (WO 92/01793); A. niger (Shei et al., 1985, Biotech, and Bio- eng. Vol. XXVII, pp. 533-538, and Fournier et al., 1985, Bio-tech. Bioeng. Vol. XXVII, pp. 539- 546; WO 91/19782 and EP 463 706); A. aculeatus (WO 94/21785).
- the xylanase is Xylanase II disclosed in WO 94/21785.
- Contemplated commercially available xylanase include SHEARZYME®, BIOFEED WHEAT®, and PULPZYMETM HC (from Novozymes A/S) and SPEZYME® CP (from Genencor Int).
- the xyloglucanases used in accordance with the invention may be any xyloglucanase, particularly of microbial origin, such as from a filamentous fungus or bacteria.
- fungal xyloglucanases include the xyloglucanases obtainable from strains belonging to the genus Malbranchea, such as the species Malbranchea cinnamomea as disclosed in WO 0112794A1 , or from strains belonging to the genus Aspergillus, such as from the species Aspergillus aculeatus as disclosed in WO 94/14953.
- bacterial xyloglucanases include the xyloglucanases obtainable from strains of the genus Paenibacillus such as the species P. pabuli or P. polymyxa as disclosed in WO 0162903A1 , or from strains of the genus Bacillus such as from the species Bacillus licheniformis and Bacillus agaradhaerens as disclosed in WO 99/02663, or from strains of the genus Jonesia.
- the cellulase used in accordance with the invention, may be any cellulase, in particular of microbial origin, in particular fungal or bacterial origin such as a cellulase derivable from a strain of a filamentous fungus (e.g., Aspergillus, Trichoderma, Humicola, Fusarium).
- a filamentous fungus e.g., Aspergillus, Trichoderma, Humicola, Fusarium.
- cellulases include the endoglucanase (endo-glucanase I) obtainable from H. insolens and further defined by the amino acid sequence of fig. 14 in WO 91/17244 and the 43 kD H. insolens endoglucanase described in WO 91/17243.
- cellulases which may be used include CELLUCLAST®, CEL- LUZYME® (available from Novozymes A/S), SPEZYME® CP (available from Genencor, USA) and ROHAMENT® 7069 W (available from Rohm, Germany).
- the pectinase may be any pectinase, in particular of microbial origin, in particular of bac- terial origin, such as a pectinase derived from a species within the genera Bacillus, Clostrid- ium, Pseudomonas, Xanthomonas and Erwinia, or of fungal origin, such as a pectinase derived from a species within the genera Aspergillus, in particular from a strain within the species A. niger and A. aculeatus.
- Contemplated commercially available pectinases include PECTI- NEX® and BIOPREPTM (available from Novozymes A/S). Enzyme activities used during saccharification or SSF
- the saccharification step or the simultaneous sac-charification and fermentation step may be carried out in the presence of a glucoamylase derived from a micro organism or a plant.
- a glucoamylase derived from a micro organism or a plant.
- glucoamylase of fungal or bacterial origin selected from the group consisting of Aspergillus niger glucoamylase, in particular A. niger G1 or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p. 1097-1102), or variants thereof, such as disclosed in WO 92/00381 and WO 00/04136; the A. awamori glucoamylase (WO 84/02921), A. oryzae (Agric.
- Aspergillus glucoamylase variants include variants to enhance the thermal stability: 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), Bio- chem. J. 301 , 275-281); disulphide bonds, A246C (Fierobe et al.
- glucoamylases include Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO 99/28448), Talaromyces leycettanus (US patent no. Re. 32,153), Talaromyces duponti, Talaromyces thermopiles (US patent no. 4,587,215).
- Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO 86/01831).
- protease(s) in the saccharification step or SST step increase(s) the FAN (Free amino nitrogen) level and increase the rate of metabolism of the yeast and further gives higher fermentation efficiency.
- Suitable proteases include fungal and bacterial proteases.
- Preferred proteases are acidic proteases, i.e., proteases characterized by the ability to hydrolyze proteins under acidic conditions below pH 7.
- Suitable acid fungal proteases include fungal proteases derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus, Endothia, Enthomophtra, Irpex, Penicillium, Sclerotiumand Toru- lopsis.
- proteases derived from Aspergillus niger see, e.g., Koaze et al., (1964), Agr. Biol. Chem. Japan, 28, 216), Aspergillus saitoi (see, e.g., Yoshida, (1954) J. Agr. Chem. Soc.
- Bacterial proteases which are not acidic proteases, include the commercially available products Alcalase® and Neutrase® (available from Novozymes A/S).
- Additional enzymes may also be used during saccharification or SSF. Additional enzymes include pullulanase and phytase.
- the micro organism may be a fungal organism, such as yeast or bacteria.
- filamentous fungi include strains of Penicillium sp.
- Preferred organisms for ethanol production is yeasts.
- Preferred yeast according to the invention is baker's yeast, also known as Saccharomyces cerevisiae.
- composition comprising an alpha-amylase, xylanase, xyloglucanase, cellulase, pectinase, and mixtures thereof.
- alpha-amylase, xylanase, cellulase, and pectinase activity may have the above-mentioned origin.
- the mash is heated to a temperature of 50-70°C, depending on the treatment.
- viscosity is measured using a Haake VT02 rotation based viscosimeter.
- the unit of viscosity is centipois (cps), which is proportionally related to the viscosity level.
- Phadebas assay Alpha-amylase activity is determined by a method employing Phadebas® tablets as substrate.
- Phadebas tablets (Phadebas® Amylase Test, supplied by Pharmacia Diagnostic) contain a cross-linked insoluble blue-colored starch polymer, which has been mixed with bovine serum albumin and a buffer substance and tabletted.
- test time is in the range of 0.2 to 2.0 absorbance units. In this absorbance range there is linearity between activity and absorbance (Lambert-Beer law). The dilution of the enzyme must therefore be adjusted to fit this criterion. Under a specified set of conditions (temperature, pH, reaction time, buffer conditions) 1 mg of a given alpha-amylase will hydrolyze a certain amount of substrate and a blue colour will be produced. The measured absorbance is directly proportional to the specific activity (activity/mg of pure alpha-amylase protein) of the alpha-amylase in question under the given set of conditions. 2.
- Alpha-amylase activity is alternatively determined by a method employing the PNP-G7 sub- strate.
- PNP-G7 which is a abbreviation for p-nitrophenyl-alpha,D-maltoheptaoside is a blocked oligosaccharide which can be cleaved by an endo-amylase.
- Kits containing PNP-G7 substrate and alpha-glucosidase is manufactured by Bohringer-Mannheim (cat. No. 1054635).
- BM 1442309 To prepare the substrate one bottle of substrate (BM 1442309) is added to 5 ml buffer (BM1442309).
- BM 1462309 To prepare the alpha-glucosidase one bottle of alpha-glucosidase (BM 1462309) is added to 45 ml buffer (BM1442309).
- the working solution is made by mixing 5 ml alpha- glucosidase solution with 0.5 ml substrate.
- the assay is performed by transforming 20 microL enzyme solution to a 96 well microtitre plate and incubating at 25°C. 200 microL working solution, 25°C is added. The solution is mixed and pre-incubated 1 minute and absorption is measured every 5 seconds over 3 minutes at OD 405 nm.
- the slope of the time dependent absorption-curve is directly proportional to the specific ac- tivity (activity per mg enzyme) of the alpha-amylase in question under the given set of conditions.
- FAU Fungal Alpha-Amylase Unit
- 5 FAU is defined as the amount of enzyme, which breaks down 5.26 g starch (Merck Amylum solubile Erg. B.6, Batch 9947275) per hour at Novozymes' standard method for determination of alpha-amylase based upon the following standard conditions: Substrate Soluble starch
- Acid alpha-amylase activity is measured in AFAU (Acid Fungal Alpha-amylase Units), which are determined relative to an enzyme standard.
- the standard used is AMG 300 L (wild type A. niger Q ⁇ AMG sold by Novozymes A/S).
- the neutral alpha-amylase in this AMG falls after storage at room temperature for 3 weeks from approx. 1 FAU/mL to below 0.05 FAU/mL.
- the acid alpha-amylase activity in this AMG standard is determined in accordance with
- AF 9 1/3 Novo method for the determination of fungal alpha-amylase.
- 1 AFAU is defined as the amount of enzyme, which degrades 5.260 mg starch dry matter per hour under standard conditions.
- Iodine forms a blue complex with starch but not with its degradation products.
- the inten- sity of colour is therefore directly proportional to the concentration of starch.
- Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under specified analytic conditions.
- the Amyloglucosidase Unit is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute at 37°C and pH 4.3.
- a detailed description of the analytical method (AEL-SM-0131) is available on request from Novozymes A/S.
- the activity is determined as AGU/ml by a method modified after (AEL-SM-0131, available on request from Novozymes A/S) using the Glucose GOD-Perid kit from Boehringer Mannheim, 124036. Standard: AMG-standard, batch 7-1195, 195 AGU/ml. 375 microL substrate (1% maltose in 50 mM Sodium acetate, pH 4.3) is incubated 5 minutes at 37°C. 25 microL enzyme diluted in sodium acetate is added. The reaction is stopped after 10 minutes by adding 100 microL 0.25 M NaOH. 20 microL is transferred to a 96 well microtitre plate and 200 microL GOD-Perid solution (124036, Boehringer Mannheim) is added. After 30 minutes at room temperature, the absorbance is measured at 650 nm and the activity calculated in AGU/ml from the AMG-standard.
- the endoxylanase activity is determined by an assay, in which the xylanase sample is incubated with a remazol-xylan substrate (4-O-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue R, Fluka), pH 6.0. The incubation is performed at 50°C for 30 min. The background of non-degraded dyed substrate is precipitated by ethanol. The remaining blue colour in the supernatant is determined spectrophotometrically at 585 nm and is proportional to the endoxylanase activity.
- a remazol-xylan substrate (4-O-methyl-D-glucurono-D-xylan dyed with Remazol Brilliant Blue R, Fluka)
- the endoxylanase activity of the sample is determined relatively to an enzyme standard.
- the assay is further described in the publication AF 293.6/1 -GB, available upon request from Novozymes A/S, Denmark.
- the xyloglucanase activity is measured using AZCL-xyloglucan from Megazyme, Ireland, (http://www.meqazyme.com/purchase/index.html) as substrate.
- a solution of 0.2 % of the blue substrate is suspended in a 0.1 M phosphate buffer pH 7.5 under stirring.
- the solution is distributed under stirring to 1.5 ml Eppendorf tubes (0J5 ml to each), 50 ⁇ l enzyme solution is added and they are incubated in an Eppendorp Thermomixer model 5436 for 20 minutes at 40°C with a mixing of 1200 rpm. After incubation the coloured solution is separated from the solid by 4 minutes centrifugation at 14,000 rpm and the absorbance of the supernatant is measured at 600 nm.
- One XyloU unit is defined as the amount of enzyme resulting in an absorbance of 0.24 in a 1 cm cuvette at 600 nm.
- ECU Endo-Glucanase Units
- the ECU endocellulose unit
- the ECU is determined relatively to an enzyme standard.
- Endocellulase decomposes carboxylmethylcellulose, CMC.
- the prepared substrate solution contains 35 g/l CMC (Blanose Aqualon) in 0.1 M phosphate buffer at pH 7.5.
- the enzyme sample to be analysed is determined is dissolved in the same buffer. 0.15 ml standard enzyme solution or the unknown enzyme sample is placed in 10 ml test tubes. 5 ml CMC-substrate isolution, preheated to 40°C, is added. The joint solution is mixed thoroughly, incubated for 30 minutes and placed in the viscometer.
- a substrate solution containing 34.0 g/l CMC (Blanose Aqualon) in 0.1 M phosphate buffer, pH 6.0 is prepared.
- the enzyme sample to be analysed is dissolved in the same buffer. 14 ml substrate solution and 0.5 ml enzyme solution are mixed and transferred to a vibration viscosimeter (e.g. MIVI 3000 available from Sofraser, France) thermostated at 40°C.
- MIVI 3000 available from Sofraser, France
- Endoglucanase unit is determined as the ratio between the viscosity of the sample and the viscosity of a standard enzyme solution.
- NCU Cellulytic Activity
- CMC carboxymethyl cellulose
- NCU One Cellulase Unit
- One Cellulase Unit is defined as the amount of enzyme which, under standard conditions (i.e., at pH 4.80; 0.1 M acetate buffer; 10 g/l Hercules CMC type 7 LFD as substrate; an incubation temp, of 40.0°C; an incubation time of 20 minutes; and an enzyme concentration of approximately 0.041 NCU/ml) forms an amount of reducing carbohydrates equivalent to 1 micro mol glucose per minute.
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Abstract
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