WO2001079483A1 - Procede de fabrication d'alcool a partir de fibre cellulosique - Google Patents

Procede de fabrication d'alcool a partir de fibre cellulosique Download PDF

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Publication number
WO2001079483A1
WO2001079483A1 PCT/JP2001/002429 JP0102429W WO0179483A1 WO 2001079483 A1 WO2001079483 A1 WO 2001079483A1 JP 0102429 W JP0102429 W JP 0102429W WO 0179483 A1 WO0179483 A1 WO 0179483A1
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Prior art keywords
microorganism
paper
cell surface
yeast
plasmid
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PCT/JP2001/002429
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English (en)
Japanese (ja)
Inventor
Hideki Fukuda
Akihiko Kondo
Yasuya Fujita
Atsuo Tanaka
Mitsuyoshi Ueda
Hideo Noda
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Kansai Chemical Engineering Co., Ltd.
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Priority to JP2001577466A priority Critical patent/JP4681199B2/ja
Publication of WO2001079483A1 publication Critical patent/WO2001079483A1/fr

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    • 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)
    • C12N9/2405—Glucanases
    • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
    • C12N9/2445—Beta-glucosidase (3.2.1.21)
    • 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
    • C12P19/02—Monosaccharides
    • 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
    • C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
    • C12P7/10—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate substrate containing cellulosic material
    • 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)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • 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)
    • C12Y302/01021—Beta-glucosidase (3.2.1.21)
    • 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)
    • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
    • 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 a method for producing a useful substance from waste generated from a paper recycling process. More specifically, the present invention relates to a method for producing alcoholic alcohol from cellulose fiber or paper which is difficult to regenerate as paper.
  • the recovered recovered paper is used as recycled paper. As the paper is recycled many times, the fibers become brittle, and some recycled paper is not used as papermaking raw material. Still, shredded paper has short fibers and is hardly a raw material in most cases. According to the above-mentioned materials, such non-recycled papers are discarded or incinerated in an amount of about ⁇ 10,000 tons, which is one of the causes of environmental destruction. Therefore, not only the recycling of paper but also how to use this discarded waste paper or paper is an important issue in reusing resources and solving environmental problems.
  • glucose can be produced from cellulose fibers or papers that have been conventionally discarded and are difficult to regenerate, and that alcohol can be produced from this glucose. Is completed.
  • the present invention relates to a method for producing glucose, which comprises a step of reacting cellulose fibers or papers, which are difficult to regenerate as paper, with an enzyme or a microorganism capable of cleaving a 1,4-darcoside bond.
  • the cellulose fibers or papers that are difficult to regenerate as paper are cellulose fibers or papers that are decomposed batchwise or continuously by a noncatalytic hydrothermal method.
  • the cellulose fiber that is difficult to regenerate as paper is a cellulose fiber decomposed by a non-catalytic hydrothermal method at 120 to 300 ° C.
  • the microorganism is capable of expressing at least one selected from the group consisting of endo] 31,4-glucanase, cellohydrolase, and j3-dalcosidase on a cell surface.
  • One or more recombinant microorganisms are provided.
  • the microorganism comprises:
  • the microorganism is yeast.
  • the present invention also provides cellulose fibers or papers that are difficult to recycle as paper, reacting an enzyme or a microorganism capable of cleaving a ⁇ 1,4-darcoside bond to produce dulcose; and reacting the obtained glucose with a microorganism B having alcohol fermentation ability. And alcohol production methods.
  • the cellulose fibers or papers which are difficult to regenerate as paper are cellulosic fibers or papers which are decomposed batchwise or continuously by a non-catalytic hydrothermal method.
  • the cellulose fiber that is difficult to regenerate as paper is a cellulose fiber decomposed by a non-catalytic hydrothermal method at 120 to 300 ° C.
  • the microorganism A may express at least one member selected from the group consisting of endo] 31,4-glucanase, cellohydrolase, and J3-dalcosidase on a cell surface.
  • One or more recombinant microorganisms may express at least one member selected from the group consisting of endo] 31,4-glucanase, cellohydrolase, and J3-dalcosidase on a cell surface.
  • said microorganism A comprises:
  • (C) A microorganism that has been recombined to express on the cell surface an enzyme selected from the group consisting of endo / 31,4-glucanase and a combination of 3-darcosidase.
  • microorganism A and the microorganism B are the same microorganism.
  • microorganism A and the microorganism B are the same yeast.
  • FIG. 1 is a diagram showing the decomposition of waste cake by cellulase.
  • FIG. 2 is a schematic diagram of the construction of plasmid pICASl.
  • FIG. 3 is a schematic diagram of the construction of plasmid pBG211.
  • FIG. 4 is a schematic diagram of the construction of plasmid pEG19.
  • FIG. 5 is a schematic diagram of the construction of plasmid pEG23u21.
  • FIG. 6 is a diagram showing the degradation of] 3-glucan using a yeast transformed with the plasmid pEG23u21 and expressing endo-1,4-glucanase on the cell surface.
  • FIG. 7 is a diagram showing the use of cellobiose by yeast expressing jS-dalcosidase 1 on the cell surface.
  • FIG. 8 is a schematic diagram showing the production of alcohol using a] 3-glucan as a substrate by using a yeast expressing ⁇ -darcosidase 1 and endo i3 1,4-gluca ⁇ ”-IIase II on the cell surface. .
  • cellulose fibers that are difficult to recycle as paper refer to wastes containing cellulose fibers generated in a paper manufacturing process or a paper recycling process, and include papermaking lees, waste paper kashiwa, and the like.
  • paper is affected by heat, humidity, light, etc. during use, and undergoes processing steps such as beating, heating, drying, and mechanical pressure in the regeneration process. Deterioration and deterioration such as damage, shortening, and hardness. For this reason, a large amount of cellulose fibers that are not made during papermaking as recycled paper are generated, and waste paper Kashiwa is generated. Cellulose fibers that are difficult to recycle as such paper are used in the present invention.
  • Paper that is difficult to recycle as paper includes, for example, paper with shortened cellulose fibers, such as shredded paper.
  • Paper is a concept that includes not only paper but also cardboard and other paperboard.
  • cellulose fibers or papers that are difficult to regenerate as the paper hereinafter, simply referred to as “raw material cellulose fibers”
  • cellulose fibers which are decomposed batchwise or continuously by a non-catalytic hydrothermal method or Paper is also preferred.
  • the raw cellulose fiber By subjecting the raw cellulose fiber to a non-catalytic hydrothermal treatment, for example, it is treated so as to form an appropriately long cellulose unit or oligosaccharide, or cross-linking between fibers (for example, between cellulose) is released, and cellulose content is reduced. It is considered that the cellulose was changed so that the decomposing enzyme could easily act.
  • the raw material cellulose fiber that has been subjected to this treatment can be used as it is as a substrate for glucose production or alcohol fermentation.
  • a decomposition product suitable for alcoholic fermentation can be obtained by treating at preferably 150 to 280 ° C, more preferably 180 to 250 ° C.
  • the processing time is preferably in the range of 1 hour to 15 seconds.
  • the hydrolysis of the raw material cellulose fiber by the non-catalytic hydrothermal method can also be performed by a continuous method.
  • a non-catalytic hydrothermal method with a slightly higher temperature may be used due to the heat history time.
  • About 10% by weight of raw material cellulose The fiber is run at 120-373 ° C, preferably 150-320 ° C, preferably for 1 hour to 1 second.
  • the raw material cellulose fiber can be converted into a decomposition product suitable for alcohol fermentation.
  • the raw material cellulose fiber is decomposed by adding, for example, sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, or the like to an acidic, preferably weakly acidic condition using a non-catalytic hydrothermal method to obtain a raw material.
  • the cellulose fiber of the family can be used as a decomposition product suitable for alcohol fermentation.
  • the “enzyme capable of cleaving a 31,4-darcoside bond” used in the present invention is not particularly limited as long as it is an enzyme capable of cleaving this ⁇ 1,4-darcoside bond. Endo] 31,4-glucanase, cellobiohydrolase, ⁇ -darcosidase, carboxymethylcellulase and the like are used.
  • a combination of two or more enzymes For example, (1) the ability to combine end j3 1,4-glucanase and ⁇ -darcosidase ⁇ (2) the ability to combine cellopiohydrolase and ⁇ -glucosidase (3) Endo j3 1,4-glucanase, cellopiohydrolase and] 3-glucosidase can be combined. It is most preferred to include
  • Microorganisms that produce enzymes that can cleave ⁇ 1,4-darcoside bonds are also preferably used. Such microorganisms include so-called cellulase producing bacteria. When referring to “cellulase", it is generally end.) 3 1, 4- Dalcanase refers to a group of enzymes that produce glucose from cellulose by cleaving the 1,4-darcoside bond produced together with endo] 31,4-dalcanase (eg, cellobiohydrolase, -dalcosidase). ) Is sometimes called cellulase. ,
  • cellulase-producing bacteria include microorganisms belonging to the genus Trichoderma, the genus Closteridium, the genus Cellus Monas, the genus Pseudomonas, and the like.
  • microorganisms that produce endo / 31 / 4-glucanase, cellobiohydrolase, and jS-darcosidase alone can also be used.
  • a microorganism that has been modified to express 4-Dulcanase on the cell surface or a microorganism that has been modified to express mouth piohydrolase on the cell surface may be used alone or in combination.
  • Examples of the combination include: a combination of endo j31,4-glucanase and ⁇ -glucosidase; and Saccharobiohydrolase; a combination of 3-dalcosidase; Endo] 31,4-glucanase; Lase and glucosidase combinations.
  • Microorganisms that are recombinant so as to express a plurality of enzymes on the cell surface are also preferably used.
  • microorganisms are the following (A) to (C):
  • Such a microorganism is created by applying a cell surface engineering technology using a so-called genetic recombination technology.
  • Murai et al., Applied and Environmental Microbiology, vol. 63, 1362-1366 are examples of cell surface engineering. According to this document, a darcoamylase gene was linked to a gene encoding 320 amino acids of the C-terminal region of ⁇ -gluture of the yeast, and dalcoamylase was immobilized on the cell surface. Disassembly.
  • Microorganisms having an enzyme that cleaves 1,4-darcoside bonds of the present invention, or host microorganisms for expressing such enzymes include, but are not limited to, filamentous fungi, bacteria, and enzymes. . Yeast is preferable in consideration of handling and the like.
  • An enzyme that cleaves a ⁇ 1,4-darcoside bond, a microorganism that produces such an enzyme, or a microorganism that expresses such an enzyme on the cell surface is preferably immobilized on a carrier. This enables reuse.
  • carrier and the method for immobilizing the enzyme those skilled in the art can use carriers and methods commonly used, and examples thereof include a carrier binding method, an entrapment method, and a cross-linking method.
  • a porous body is preferably used as a carrier for immobilizing microorganisms.
  • a foam or resin such as polyvinyl alcohol, polyurethane foam, polystyrene foam, polyacrylamide, porous polyformal resin, and silicon foam is preferable.
  • the size of the opening of the porous body may be determined in consideration of the size of the microorganism used. In the case of yeast, 50 to 1000 ⁇ is preferred.
  • the shape of the carrier is not limited. Considering the strength of the carrier, cultivation efficiency, etc., Spherical or cubic are preferred.
  • the size may be determined depending on the microorganism to be used. In general, the diameter is preferably 2 to 50 m;
  • Glucose is produced when the above enzymes, microorganisms, immobilized enzymes, and immobilized microorganism are reacted with the cellulosic fiber of the raw material.
  • concentration of a raw material (cellulose fiber as raw material) serving as a substrate for the enzyme is not particularly limited.
  • the reaction is carried out at an appropriate temperature (for example, generally 10 to 70 ° C) for an appropriate time depending on the enzyme used.
  • the reaction can proceed at 90 ° C. or higher.
  • This reaction can be a continuous reaction using a column when immobilized enzymes or microorganisms are used. Endo 1,4-Dulcanase, cellohydrohydrolase, and 3-Darcosidase treatments can be performed in this order by multistage column treatment. The obtained glucose is isolated by a conventional method.
  • Production of alcohol from glucose is also one of the present inventions. Using the obtained glucose as a substrate, a microorganism capable of alcohol fermentation may be reacted.
  • the microorganism capable of alcohol fermentation is not particularly limited, but yeast is preferably used.
  • the yeast capable of alcohol fermentation is not particularly limited, and includes yeasts conventionally used in the fermentation industry, such as sake yeast, brewer's yeast, wine yeast, and baker's yeast.
  • alcoholic fermentation In alcoholic fermentation, a two-step reaction is generally considered, in which darcos is first produced from the raw material cellulose fiber, and yeast having alcoholic fermentation ability is added thereto.
  • Another method is to perform alcohol fermentation directly from the raw cellulose fiber.
  • This includes (i) a method using a decomposition solution obtained by decomposing the raw material cellulose fiber by a non-catalytic hydrothermal method, and (ii) decomposition of the raw material cellulose fiber and alcohol. And (iii) simultaneous decomposition of cellulose fibers in a decomposition solution obtained by decomposing the raw material cellulose fibers by a non-catalytic hydrothermal method and alcohol fermentation.
  • the decomposition liquid obtained by decomposing the raw material cellulose fiber by the non-catalytic hydrothermal method can be used as it is as the raw material for alcohol fermentation. It is performed by adding yeast having a function.
  • the method (ii) is carried out by coexisting an enzyme capable of decomposing cellulose fibers as a raw material or a (yet-replaced) microorganism and a microorganism having an alcohol fermentation ability.
  • a yeast having an alcohol fermentation ability can be used as a host, for example, by using a yeast that has been modified to express ⁇ -glucosidase and ⁇ or end 3,4-glucanase on the cell surface. , Done.
  • this recombinant yeast is used, the steps of decomposing the cellulose fibers to produce glucose and the step of fermenting the produced glucose with alcohol are performed simultaneously, which is effective.
  • the method (iii) is a combination of the methods (i) and (ii), and is a method for producing alcohol by further effectively utilizing the raw cellulose fiber.
  • Such a microorganism having an alcohol fermentation ability may be immobilized as described above for glucose production.
  • Example 1 Glucose production from cellulose fiber that is difficult to regenerate
  • the waste liquid from the recycled paper manufacturing process was collected, and the unregenerated cellulose fibers were recovered.
  • the recovered cellulose fibers are hereinafter referred to as used paper meal.
  • crystalline Abysse (Avicel) and Filter paper powder C were prepared. These are dispersed in a 0.1 M sodium acetate buffer to a concentration of lOg / L, and reacted at 30 ° C using cellulase derived from Trichoderma reesei (manufactured by Funakoshi Co., Ltd.) to produce glucose. Quantification was performed using CII Test II Co. (Wako Pure Chemical Industries, Ltd.). The results are shown in Figure 1.
  • ⁇ represents waste paper meal
  • ⁇ represents Avicel
  • ⁇ represents Filter paper powder r C, respectively.
  • Figure 1 shows for the first time that waste paper meal is degraded much more efficiently than other cellulosic materials, indicating that waste paper kashiwa is extremely promising as a raw material for glucose production. I have.
  • Example 2 Alcohol production from waste paper meal using yeast expressing ⁇ -darcosidase 1 and / or end 1,4-glucanase I on the cell surface
  • FIGS. 2 and 3 show schematic diagrams for preparing a plasmid that expresses j3-dalcosidase 1 on the cell surface.
  • Figure 2 is a schematic diagram of the creation of the plasmid pICASl, which is the material for the target plasmid, pBG211.
  • a plasmid pYGA2269 (Ashikari et al., Appl. Microbiol. Biotechnol. 30: 515-520 (1989)) having a sequence in which a DAP-coamylase derived from Rhizopus oryzae is connected to GAPDH promoter 1 to goo from baccharomyces cerevisiae Two oligonucleotides 5, -ccgagctcaccagttctcaccggaaca-3
  • Plasmid pGAll (Murai et al., Appl Environ Microbiol 63:.. . 1362- 1366 (1997)) cut out Xhol- Kpnl fragment from, DNA fragment containing the C-terminal 320 amino acids and 44 [rho flanking region of a single Aguruchun shed (Fragment II I) was obtained.
  • plasmid pRS404 (Sikorski et al., Genetics 122: 19-17 (1989)) was cut with Sacl-Kpnl, and the above fragments I to [II were mixed to produce plasmid pICAS1.
  • This plasmid has a sequence containing the GAPDH promoter, a secretory sequence, the C-terminal 320 amino acids of a-agglutinin, and a 446 bp flanking region in this order.
  • FIG. 3 shows the procedure for creating the desired plasmid pBG211 using this plasmid pICASl.
  • Plasmid pICASl was digested with Bglll-Xhol, and the jS-darcosidase 1 gene from Aspergillus acule atus of plasmid pABG7 (Kawaguchi et al., Gene 173: 287-288 (1996)) was inserted into this site.
  • DNA was obtained by PCR using plasmid pABG7 as a template, 5, _gtcgagatctctga1: gaactggcgttctct-3, (distribution system's J number 5 no and -ttcactcgagccttgcaccttcgggagcgccg-3 '(measure ⁇ 1> lj ⁇ " ⁇ 6) as a primer.
  • This fragment was digested with Bglll-Xhol, and this fragment was introduced into the Bglll-Xho cleavage site of the plasmid pICASl to obtain a plasmid pCS.
  • This plasmid pMHCS contains the GAPDH promoter and secretory signal. [J, i3 -.
  • alpha-Aguruchun has a gene sequence comprising the C-terminal region 320 amino acids and 44 6 bp flanking regions pMHCS was digested with BssH II and, GAPDH promoter, a secretory signal sequence BssHII-BssHII DNA fragment (320 amino acids and 446 bp flanking region) of the ⁇ -agglutin C-terminal region and the ⁇ -dalcosidase structural gene Segment IV) was isolate.
  • plasmid pMT34 (+3) containing 2 imDNA (Tajima et al., Yeast 1: 67 7 7 (1985)), and inserted into the Aatll site of plasmid pRS403 (Sikorski et al., Genetics 122: 19-17 (1989)) to produce multicopy plasmid pMHl.
  • This plasmid pffll was digested with BssHII, and fragment IV was inserted therein to obtain a plasmid pBG211 expressing -dalcosidase on the cell surface.
  • Fuhus 3 ⁇ 4> pBG21 ⁇ ⁇ 3 ⁇ 4 ⁇ 3 ⁇ 43 ( ⁇ (; ⁇ 3 ⁇ 3 cerevisiae MT81-1 (MATa ura3 trpl a de leu2 his3)) was introduced by the lithium acetate method using Yeast Maker (manufactured by CLONTEC).
  • Medium (6.7 g / L yeast nitrogen base w / h amino acids, 20 g / L gnorecose, 0.03 g / L leucine, 0.02 g / L tryptophan, 0.02 g / L adenine, 0.02 g / L peracil). and the transformant was designated as MT8- 1 / P BG211.
  • Figure 4 shows a schematic diagram of the construction of plasmid pEG19 that expresses endoglucanase I on the cell surface.
  • the EcoRI DNA fragment of plasmid pWI3 with 2 mDNA was converted to the plasmid pRS405 (Sikorski et al., Genetics 122: 19-17 (1989)).
  • the plasmid was introduced into the Aatll site to generate plasmid pRS405 + 2.
  • GAPDH promoter of the plasmid Picas l, secretion signal sequence, a DNA fragment containing the C-terminal 320 Amino acid and 446bp flanking region of ⁇ - Aguruchinin was amplified by PCR, was cut with EcoRV, a plasmid P RS405 + The plasmid was introduced into the Pvul I-Pvu II site of No. 2 to prepare a plasmid pMLCS5.
  • the primer used for amplification was 5-ggaaacagctatgaccatgatatcgccaagcgcgcaa.tta-3 '(Rooster column number 7)
  • the endo j31,4-glucanase gene derived from Tricoderma reesei was inserted into the Bglll site of this plasmid pMLCS5. That is, as the 1st strana cDNA unplate prepared from the mRNA of T.
  • the obtained plasmid pEG19 was transformed with the yeast Saccharomyces cerevisiae MT—8 in the same manner as in 2-1 to obtain SDi3 ⁇ 4 "i-ya (6.7 g / L yeast nitrogen base w / o amino acids, 20 g / L (Gnorecose, 0.02 g / L tryptophan, 0.02 g / L histidine, 0.02 g / L adenine, 0.02 g / L peracil)
  • the obtained transformant was designated as MT8-l / pEG19.
  • plasmid pEG19 was transformed in the same manner as 2-1.SDJ3 ⁇ 4-3 ⁇ 4 (6.7 g / L yeast nitrogen base w / o amino acids, 20 g / L gnorecose, .02 g / L tryptophan, 0.02 g / L adenine, and 0.02 g / L peracil).
  • the resulting transformant was designated as MT8_1 / pBG211 + pEG19.
  • a transformant (MT8-l / pBG211 + pEG19) that expresses 3-dalcosidase 1 and endoglucanase I on the cell surface is suspended in the SD medium (liquid medium) used for each selection, and then suspended at 30 ° C. C, and cultured for 48 hours to obtain a pre-culture solution.
  • This pre-cultured solution was used as a 2 L jar armmenter (BMJ-02PIAb le) containing 1 L of YPD medium (10 g / L yeast extract, 20 g / L polypeptide, 5 g / L of glucose) containing 40 g / L waste paper meal. ) And cultured under microaerobic conditions of 0.01 to 0.03 ppm. All transformants grew despite the small amount of glucose added. This is probably because glucose was produced by the enzyme expressed on the cell surface, and it grew using the produced darcose. / 3 - Darukoshidaze growth 1 and Endodaru 'Kanaze I and a transformant that expressed on the cell surface (MT8-l / P BG211 + pEG19) was the best.
  • the culture was terminated, the cells were collected by centrifugation, and 5 g / LYPD medium containing 70 g / L waste paper meal was used at 30 ° C, pH 6.0, Fermentation was performed under microaerobic conditions of 0.01 to 0.03 ppm.
  • the results of measuring the ethanol in the culture broth showed that the transformant (MT8-l / pBG211), the transformant (MT8-1 / PEG19) and the transformant (MT8-l / pBG211 + pEG19) ) Produced 3 g / L, 4 g / L and 6 g / L ethanol, respectively.
  • the numbers represent the alcohol concentration (g / s) .
  • Table 1 show that when the waste paper treated at 400 ° C for 15 seconds is added to the culture medium compared to when untreated waste paper is added to the culture medium, the alcohol fermentation The degree was small, and it was even lower when mashed at 400 ° C for 30 minutes. The degree of alcohol fermentation was much greater when waste paper treated under milder conditions was added than without. It is probable that alcohol fermentation did not proceed due to factors such as the decomposition of cellulose to produce the necessary sugars or the formation of substances that hinder the reaction due to the treatment of waste paper under harsh conditions.
  • Example 3 Construction of yeast expressing endo / 3 1,4-glucanase II on cell surface and degradation of glucan
  • endoglucanase II Preparation of yeast that expresses 3-1 endo
  • Figure 5 shows the procedure for preparing plasmid pEG23u31 that expresses endoglucanase II on the cell surface.
  • the plasmid pURI24 created by the present inventors Tanaka and Ueda, was cut with PvuII and BamHI to obtain blunt ends.
  • An EcoRV-EcoRV fragment was inserted into this blunt-ended site to create a plasmid pMUCS.
  • PCR was carried out using the mRNA of Trichoderma reesei as a template and the prepared 1st strand cDNA.
  • the primers used were 5, -cggcgagatctcacagcaga ctgtctgggg-3 '(rooster column number) and D -gacagctcgagggctttcttgcgagacacg-3' (SEQ ID NO: 12).
  • the PCR product was cut with Bglll and Xhol, and A Bgll I-Xhol fragment having a sequence encoding 4-gunolecanase II (glucanase II) was obtained, and the Bglll-Xhol fragment was inserted into the Bglll-Xhol site of plasmid pMUCS to obtain the desired plasmid pEG23u31. .
  • the obtained plasmid pEG23u31 was introduced into the yeast Saccharomyces cerevisiae MT-8 in the same manner as 2-1 and SD ⁇ ya (6.7 g / L yeast nitrogen base w / o ami no acids, 20 g / L glucose). , 0.02 g / L tryptophan, 0.02 g / L histidine, 0.02 g / L adenine, and 0.03 g / L leucine.
  • the resulting transformant was designated as MT8-l / pEG23u31.
  • Example 4 Preparation of yeast expressing 3 dalcosidase 1 and ⁇ or endoglucanase II on the cell surface
  • yeast transformed with a chromosomal integration plasmid was prepared.
  • PCR was performed using pBG211 prepared in 2-1 as a template to obtain a PCR product having a sequence encoding ⁇ -glucosidase 1.
  • the primers used were D-gatctccatggctgatgaactggcgttctctcctcttttc-3 (Rokki No. 13) and 5-tggcgctcgagccttgcaccttcgggagcgccgcgtgaag-3 '(Rokki U number 14).
  • the obtained PCR product was digested with Ncol and Xhol, introduced into the Ncol-Xhoi site of plasmid pIHCS, and the desired plasmid,] 3-dalcosidase 1 surface expression, chromosome-integrated plasmid pIBG13 was prepared. .
  • the obtained plasmid PIBG13 is cut into single-stranded DNA by cutting with Nhel, and then introduced into yeast Saccharomyces cerevisiae MT-8 in the same manner as in 2-1 to obtain an SD medium (6.7 g / h yeast).
  • an SD medium 6 g / h yeast.
  • the transformant was designated as MT8-1 / pIBG13.
  • Endalcanase II was performed by incubating at 30 ° C for 48 hours on an SD selection plate containing lg / L-glucan and staining with lg / L Congo Red to confirm halo.
  • the transformant MT8-l / pEG23u31pIBG13 was transferred to an SD medium (6.7 g / L yeast nitrogen base w / o amino acids, 20 g / L gnorecose, 0.03 g / L leucine, 0.02 g / L triptofan, 0.02 g / L-Adenine, pre-cultured in a medium of 0.02 g / L peracil, and cultured on SD medium (6.7 g / L yeast nitrogen base w / o amino acids ⁇ lOg / L j3-glucan,
  • the cells were cultured with 03 g / L leucine, 0.02 g / L tryptophan, 0.02 g / L adenine, and 0.02 g / L ⁇ racil), and the amount of ethanol formed was measured by gas chromatography. Total sugars were measured by the sorbazole-sulfuric acid method. Fig. 8 shows the results. This result indicates that] -glucan was completely saccharified and alcohol fermentation was performed. Industrial applicability
  • cellulose fibers that are not recycled as paper are effectively reused as resources.
  • a microorganism that has been subjected to an enzyme capable of cleaving a 1,4-darcoside bond is preferably used.

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Abstract

L'invention concerne des fibres cellulosiques ou du papier, que l'on peut difficilement recycler en papier, et que l'on fait réagir avec une enzyme ou un microorganisme capable d'induire le clivage d'une liaison β-1,4-glucoside, de manière à produire du glucose. Ensuite, la fermentation de l'alcool intervient, ce qui permet d'utiliser efficacement la ressource papier qui a été jetée. On peut ainsi produire du glucose, puis de l'alcool, dans des conditions particulièrement efficaces en utilisant un microorganisme qui exprime sur la couche de surface cellulaire au moins une enzyme pouvant être β-1,4-glucanase, cellobiohydrolase et β-glucoside.
PCT/JP2001/002429 2000-04-17 2001-03-26 Procede de fabrication d'alcool a partir de fibre cellulosique WO2001079483A1 (fr)

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KR100482192B1 (ko) * 2002-09-03 2005-04-13 학교법인 인하학원 제지 슬러지로부터 젖산을 생산하는 방법
JP2007259803A (ja) * 2006-03-29 2007-10-11 Toyota Central Res & Dev Lab Inc β―グルコシダーゼ活性を有するタンパク質及びその利用
JP2008514391A (ja) * 2004-09-24 2008-05-08 キャンビ・バイオエタノール・アンパルトセルスカブ 望ましい生物系産物を生成することを目的としてバイオマスおよび有機廃棄物を処理する方法
CN100400550C (zh) * 2004-10-13 2008-07-09 姜国文 生物肽重烃解离酶双氢转因子改性纤维素的制备方法
JP2008193935A (ja) * 2007-02-09 2008-08-28 Ｂｉｏ−ｅｎｅｒｇｙ株式会社 エタノールの製造方法
JP2009033993A (ja) * 2007-07-31 2009-02-19 Toyota Central R&D Labs Inc セルラーゼ担持材料及びその利用
JP2009112200A (ja) * 2007-11-02 2009-05-28 Nippon Steel Engineering Co Ltd エタノール製造方法
WO2009139349A1 (fr) * 2008-05-14 2009-11-19 Ｂｉｏ－ｅｎｅｒｇｙ株式会社 Procédé d’introduction d’un gène dans une cellule de levure et vecteur pour le procédé
JP2010538642A (ja) * 2007-09-12 2010-12-16 マーテック バイオサイエンシーズ コーポレーション 生物油ならびにその生産および使用
WO2011067960A1 (fr) * 2009-12-01 2011-06-09 Ｂｉｏ－ｅｎｅｒｇｙ株式会社 Procédé de production d'éthanol
US7960511B2 (en) 2008-04-10 2011-06-14 Kabushiki Kaisha Toyota Chuo Kenkyusho Acid-resistance endoglucanase and the use of thereof
JPWO2010101158A1 (ja) * 2009-03-02 2012-09-10 住友商事株式会社 クロストリジウムセルロボランス由来新規遺伝子及びその利用
US8557586B2 (en) 2010-12-10 2013-10-15 National University Corporation Kobe University Cellulose degradable yeast and method for production thereof
US8574911B2 (en) 2008-09-17 2013-11-05 Kansai Chemical Engineering Co., Ltd. Production and use of yeast having increased cellulose hydrolysis ability
WO2018131653A1 (fr) * 2017-01-12 2018-07-19 新日鉄住金エンジニアリング株式会社 Procédé et appareil de production d'une enzyme de saccharification pour la saccharification de biomasse lignocellulosique, et utilisations desdits procédé et appareil

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KR100482192B1 (ko) * 2002-09-03 2005-04-13 학교법인 인하학원 제지 슬러지로부터 젖산을 생산하는 방법
JP4722932B2 (ja) * 2004-09-24 2011-07-13 キャンビ・バイオエタノール・アンパルトセルスカブ 望ましい生物系産物を生成することを目的としてバイオマスおよび有機廃棄物を処理する方法
JP2008514391A (ja) * 2004-09-24 2008-05-08 キャンビ・バイオエタノール・アンパルトセルスカブ 望ましい生物系産物を生成することを目的としてバイオマスおよび有機廃棄物を処理する方法
CN100400550C (zh) * 2004-10-13 2008-07-09 姜国文 生物肽重烃解离酶双氢转因子改性纤维素的制备方法
JP2007259803A (ja) * 2006-03-29 2007-10-11 Toyota Central Res & Dev Lab Inc β―グルコシダーゼ活性を有するタンパク質及びその利用
JP2008193935A (ja) * 2007-02-09 2008-08-28 Ｂｉｏ−ｅｎｅｒｇｙ株式会社 エタノールの製造方法
JP2009033993A (ja) * 2007-07-31 2009-02-19 Toyota Central R&D Labs Inc セルラーゼ担持材料及びその利用
US9453172B2 (en) 2007-09-12 2016-09-27 Dsm Ip Assets B.V. Biological oils and production and uses thereof
JP2010538642A (ja) * 2007-09-12 2010-12-16 マーテック バイオサイエンシーズ コーポレーション 生物油ならびにその生産および使用
JP2015002743A (ja) * 2007-09-12 2015-01-08 ディーエスエム アイピー アセッツ ビー．ブイ． 生物油ならびにその生産および使用
JP2009112200A (ja) * 2007-11-02 2009-05-28 Nippon Steel Engineering Co Ltd エタノール製造方法
US7960511B2 (en) 2008-04-10 2011-06-14 Kabushiki Kaisha Toyota Chuo Kenkyusho Acid-resistance endoglucanase and the use of thereof
JPWO2009139349A1 (ja) * 2008-05-14 2011-09-22 Ｂｉｏ−ｅｎｅｒｇｙ株式会社 酵母細胞に遺伝子を導入する方法およびそのためのベクター
WO2009139349A1 (fr) * 2008-05-14 2009-11-19 Ｂｉｏ－ｅｎｅｒｇｙ株式会社 Procédé d’introduction d’un gène dans une cellule de levure et vecteur pour le procédé
US8574911B2 (en) 2008-09-17 2013-11-05 Kansai Chemical Engineering Co., Ltd. Production and use of yeast having increased cellulose hydrolysis ability
JPWO2010101158A1 (ja) * 2009-03-02 2012-09-10 住友商事株式会社 クロストリジウムセルロボランス由来新規遺伝子及びその利用
WO2011067960A1 (fr) * 2009-12-01 2011-06-09 Ｂｉｏ－ｅｎｅｒｇｙ株式会社 Procédé de production d'éthanol
JP5752049B2 (ja) * 2009-12-01 2015-07-22 Ｂｉｏ−ｅｎｅｒｇｙ株式会社 エタノールの製造方法
US8557586B2 (en) 2010-12-10 2013-10-15 National University Corporation Kobe University Cellulose degradable yeast and method for production thereof
WO2018131653A1 (fr) * 2017-01-12 2018-07-19 新日鉄住金エンジニアリング株式会社 Procédé et appareil de production d'une enzyme de saccharification pour la saccharification de biomasse lignocellulosique, et utilisations desdits procédé et appareil

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