WO2015099109A1 - 糖液の製造方法 - Google Patents
糖液の製造方法 Download PDFInfo
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- WO2015099109A1 WO2015099109A1 PCT/JP2014/084476 JP2014084476W WO2015099109A1 WO 2015099109 A1 WO2015099109 A1 WO 2015099109A1 JP 2014084476 W JP2014084476 W JP 2014084476W WO 2015099109 A1 WO2015099109 A1 WO 2015099109A1
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- cellulose
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- enzyme
- containing biomass
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- 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
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- 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
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- 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 sugar solution that can be used as a fermentation raw material from cellulose-containing biomass.
- the fermentation production process of chemicals using sugar as a raw material is used for the production of various industrial raw materials.
- sugar derived from edible raw materials such as sugar cane, starch and sugar beet is used industrially as sugar for this fermentation raw material, but the price of edible raw materials will rise due to the increase in the world population in the future, or it will compete with edible foods.
- a process for producing sugar solution more efficiently than renewable non-edible resources, ie cellulose-containing biomass, or a process for efficiently converting the obtained sugar solution as a fermentation raw material into an industrial raw material The future is a future challenge.
- Non-Patent Document 1 a method for producing a sugar solution by treating a cellulose-containing biomass with subcritical water at about 250 to 500 ° C.
- Patent Document 1 A method (Patent Document 2) is disclosed in which a sugar solution is produced by hydrolyzing with pressurized hot water at 240 to 280 ° C. and then further with a saccharifying enzyme treatment.
- Patent Document 2 A method for hydrolyzing biomass using a saccharifying enzyme that has a particularly low energy consumption and an environmental load and a high sugar yield has been widely studied. There was a problem of high costs.
- JP 2001-95597 A Japanese Patent No. 3041380 JP 2006-87319 A JP-A-63-87994
- the present invention has the following configurations [1] to [7].
- [1] A method for producing a sugar solution from cellulose-containing biomass, The cellulose-containing biomass is hydrolyzed with filamentous fungus-derived cellulase (1) and the hydrolyzate obtained in step (1) is filtered through an ultrafiltration membrane, and the filamentous fungus-derived cellulase is recovered as a non-permeate.
- Including a step (2) of obtaining a sugar solution as a permeate A method for producing a sugar solution, comprising treating cellulose-containing biomass with one or more enzymes selected from the group consisting of pectinase, glucoamylase and lipase in the previous stage of step (1) or in step (1).
- the cellulose-containing biomass is treated with any of three enzymes, pectinase, glucoamylase, and lipase, in the preceding stage of the step (1) or in the step (1), according to any one of [1] to [4] Method for producing a sugar solution.
- the step (2) is a step of filtering the solution component obtained by solid-liquid separation of the hydrolyzate obtained in the step (1) through an ultrafiltration membrane, from [1] [5] The method for producing a sugar liquid according to any one of [5].
- Step (1) is a step of hydrolyzing cellulose-containing biomass with a filamentous fungus-derived cellulase.
- the cellulose-containing biomass used in step (1) refers to a biological resource containing at least cellulose.
- cellulose-containing biomass include herbaceous biomass such as bagasse, switchgrass, napiergrass, Eliansus, corn stover, straw (rice straw, wheat straw), or woody biomass such as trees and waste building materials, algae, Examples include biomass derived from the aquatic environment such as seaweed, and cereal skin biomass such as corn husk, wheat husk, soybean hull, rice husk, etc., but are most effective and preferably used for cereal skin biomass, straw and bagasse.
- the hydrolysis of cellulose-containing biomass is intended to reduce the molecular weight of cellulose to produce monosaccharides or oligosaccharides.
- hemicellulose components such as xylan, mannan, and arabinan are simultaneously hydrolyzed.
- a filamentous fungus-derived cellulase is used as a saccharifying enzyme to hydrolyze cellulose-containing biomass.
- the filamentous fungus-derived cellulase is an enzyme composition having a plurality of enzyme components such as cellobiohydrolase, endoglucanase, exoglucanase, ⁇ -glucosidase, xylanase, and xylosidase and having an activity of hydrolyzing and saccharifying cellulose.
- the filamentous fungus-derived cellulase can efficiently hydrolyze cellulose due to the concerted effect or complementary effect of a plurality of enzyme components in cellulose degradation.
- the cellulase derived from filamentous fungi the genus Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Amicula, Humol, ), Irpex, Mucor, Talaromyces, Phanerochaete, white rot fungus, brown rot fungus, and the like.
- filamentous fungus-derived cellulases it is preferable to use a Trichoderma-derived cellulase having a high cellulose-degrading activity.
- Trichoderma-derived cellulase is an enzyme composition mainly composed of cellulase derived from Trichoderma microorganisms.
- the microorganism of the genus Trichoderma is not particularly limited, but Trichoderma reesei (Trichoderma reesei) is preferable, and specifically, Trichoderma reesei QM9414 (Trichoderma reesei QM9414), Trichoderma reesei QM9123 (Trichoderma reesei QM9123) reeseiRut C-30), Trichoderma reesei PC3-7 (Trichoderma reesei PC3-7), Trichoderma reesei CL-847 (Trichoderma reeseiCL-847), Trichoderma reesei MCG77 (Trichoderma MCe 77) Ma reesei MCG80 (Trichoderma reeseiMCG80), can be exempl
- Cellobiohydrolase is a general term for enzymes characterized by hydrolysis from the terminal portion of cellulose in cellobiose units.
- Cellobiohydrolase I which is cleaved from the reducing end of the cellulose chain
- non-cellobiohydrolase I Two types of cellobiohydrolase II that are cleaved from the reducing end are known.
- An enzyme group belonging to cellobiohydrolase is described as EC number: EC 3.2.1.91.
- Endoglucanase is a general term for enzymes characterized by random hydrolysis from the central part of the cellulose molecular chain.
- EC numbers: EC 3.2.1.4, EC 3.2.1.6, EC 3.2 Enzymes belonging to endoglucanase are described as 1.3.9 and EC 3.2.1.73.
- Exoglucanase is a general term for enzymes characterized by random hydrolysis from the end of cellulose molecular chain, and it belongs to exoglucanase as EC numbers: EC3.2.1.74 and EC3.2.1.58. Enzymes are described.
- ⁇ -glucosidase is a general term for enzymes characterized by acting on cellooligosaccharide or cellobiose, and an enzyme group belonging to ⁇ -glucosidase is described as EC number: EC 3.2.1.21.
- Xylanase is a general term for enzymes characterized by acting on hemicellulose or especially xylan, and an enzyme group belonging to xylanase is described as EC number: EC3.2.1.8.
- Xylosidase is a general term for enzymes characterized by acting on xylo-oligosaccharides, and an enzyme group belonging to xylosidase is described as EC number: EC 3.2.1.37.
- a crude enzyme product is preferably used as the filamentous fungus-derived cellulase.
- the crude enzyme product is derived from the culture supernatant obtained by culturing the microorganism for an arbitrary period in a medium prepared so that the filamentous fungus produces cellulase.
- the medium components to be used are not particularly limited, but in order to promote the production of cellulase, a medium to which cellulose is added can be generally used.
- the culture supernatant is preferably used as it is, or the culture supernatant from which the cells have been removed.
- the weight ratio of each enzyme component in the crude enzyme product is not particularly limited.
- the culture solution derived from Trichoderma reesei contains 50 to 95% by weight of cellobiohydrolase, and the rest.
- the endoglucanase, ⁇ -glucosidase and the like are included in the components.
- Trichoderma microorganisms produce strong cellulase components in the culture solution, while ⁇ -glucosidase is retained in the cell or on the cell surface and therefore has low ⁇ -glucosidase activity in the culture solution.
- a heterogeneous or homologous ⁇ -glucosidase may be further added to the enzyme product.
- ⁇ -glucosidase derived from Aspergillus can be preferably used.
- ⁇ -glucosidase derived from the genus Aspergillus include “Novozyme 188” commercially available from Novozyme.
- a method of adding a heterologous or homologous ⁇ -glucosidase to a crude enzyme product a gene is introduced into a Trichoderma microorganism, and the Trichoderma microorganism that has been genetically modified so as to be produced in the culture solution is cultured. A method of isolating the culture solution may also be used.
- the pretreatment method for cellulose-containing biomass is not particularly limited. Specifically, acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkali treatment, caustic soda treatment, ammonia treatment, hydrothermal treatment, subcritical water treatment, pulverization treatment, steaming In the present invention, hydrothermal treatment and dilute sulfuric acid treatment are preferred.
- Hydrothermal treatment is a treatment for 1 second to 60 minutes at a temperature of 100 to 400 ° C. after adding water so that the biomass solids concentration is 0.1 to 50% by weight.
- the temperature is preferably in the range of 100 ° C. to 250 ° C.
- the treatment time is preferably 5 minutes to 30 minutes.
- the number of processes is not particularly limited, and the process may be performed once or more. In particular, when the process is performed twice or more, the first process and the second and subsequent processes may be performed under different conditions.
- dilute sulfuric acid treatment may be performed by adding sulfuric acid in hydrothermal treatment.
- the amount of sulfuric acid added is preferably 0.1 to 150 mg of sulfuric acid per 1 g of cellulose-containing biomass.
- the reaction conditions for the hydrolysis by the filamentous fungus-derived cellulase are not limited as long as they are carried out in accordance with the preferred reaction conditions for the filamentous fungus-derived cellulase, but the general reaction temperature when using the filamentous fungus-derived cellulase is 15-100 ° C.
- the range is preferably 40 to 60 ° C, more preferably 50 ° C.
- the pH of hydrolysis is preferably in the range of pH 3 to 9, more preferably pH 4 to 5.5, and even more preferably pH 5.
- acid or alkali can be added and adjusted so as to have a desired pH, and a buffer solution may be used as appropriate.
- the solid content concentration of cellulose is preferably 1 to 25% by weight. More preferably, the water is added in a range of 5 to 20% by weight.
- Step (2) the hydrolyzate obtained in the step (1) is filtered through an ultrafiltration membrane, the filamentous fungus-derived cellulase is recovered as a non-permeate, and a sugar solution is obtained as a permeate.
- the enzyme recovered as a non-permeate can be reused in step (1), and the amount of enzyme used in step (1) can be reduced.
- the molecular weight cutoff of the ultrafiltration membrane used in the present invention is particularly an ultrafiltration membrane that can permeate at least monosaccharides such as glucose (molecular weight 180) and xylose (molecular weight 150), and can inhibit filamentous fungus-derived cellulase.
- the molecular weight cut-off is preferably in the range of 500 to 50,000, and more preferably a molecular weight cut-off of 5 from the viewpoint of separating contaminants having an inhibitory action on the enzyme reaction from the enzyme. In the range of 10,000 to 50,000, and more preferably in the range of 10,000 to 30,000.
- Ultrafiltration membrane materials include polyethersulfone (PES), polysulfone (PS), polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), regenerated cellulose, cellulose, cellulose ester, sulfonated polysulfone, and sulfonated polyether.
- PES polyethersulfone
- PS polysulfone
- PAN polyacrylonitrile
- PVDF polyvinylidene fluoride
- regenerated cellulose, cellulose, cellulose ester polyvinylidene fluoride
- regenerated cellulose, cellulose, cellulose ester polymethyl methacrylate
- polytetrafluoroethylene etc.
- synthetic polymers such as PES and PVDF are used as materials. It is preferable to use an ultrafiltration membrane as described above.
- the filtration method of the ultrafiltration membrane there are dead-end filtration and crossflow filtration. From the viewpoint of suppressing membrane fouling, crossflow filtration is preferable.
- an appropriate form such as a flat membrane type, a spiral type, a tubular type, and a hollow fiber type can be used.
- the present invention is characterized in that the cellulose-containing biomass is treated with one or more enzymes selected from the group consisting of pectinase, glucoamylase and lipase in the previous stage of step (1) or in step (1).
- step ( 2) by treating cellulose-containing biomass with one or more enzymes selected from the group consisting of pectinase, glucoamylase and lipase, the step ( 2)
- the effect that the amount of filamentous fungus-derived cellulase that can be recovered is increased, and among the enzyme components contained in the filamentous fungus-derived cellulase, cello, which is the main enzyme component in hydrolyzing cellulose-containing biomass, is obtained.
- the effect of significantly increasing the amount of biohydrolase recovered is obtained.
- Pectinase refers to an enzyme having an activity of degrading pectin.
- Pectin is a complex polysaccharide that constitutes a plant body, and is a polysaccharide mainly composed of polygalacturonic acid in which galacturonic acid is ⁇ -1,4 linked.
- the pectinases include polygalactoclonase (EC 3.2.1.15), pectin lyase (EC 4.2.2.10), pectate lyase (EC 4.2.2.2), pectate methyl esterase (EC 3. 1.1.11) is a definition including at least an enzyme species related to pectin hydrolysis.
- Glucoamylase (EC 3.2.1.3) refers to an enzyme having an activity of hydrolyzing the ⁇ 1,4 bond at the non-reducing end of starch.
- Lipase refers to an enzyme that hydrolyzes lipids, and particularly has an activity to hydrolyze the ester bond between fatty acid and triglyceride (EC 3.1.1.3).
- the pectinase, glucoamylase or lipase used in the present invention is preferably derived from the genus Aspergillus.
- the genus Aspergillus include Aspergillus nigar, Aspergillus oryzae, Aspergillus awamori, Aspergillus acretus (Aspergils, etc.).
- Aspergillus-derived enzymes have an optimum reaction temperature at which the enzyme activity is maximized at around 50 ° C., and can be used at the same temperature when used in combination with filamentous fungus-derived cellulases as described later.
- the treatment of cellulose-containing biomass with one or more enzymes selected from the group consisting of pectinase, glucoamylase and lipase in the previous stage of step (1) or in step (1) is specifically the cellulose in step (1).
- the contained biomass is hydrolyzed with at least one enzyme selected from the group consisting of pectinase, glucoamylase and lipase before or during hydrolysis by the filamentous fungus-derived cellulase in step (1).
- a step of hydrolyzing the hydrolyzate obtained in step (1) with one or more enzymes selected from the group consisting of pectinase, glucoamylase and lipase are also included.
- One or more enzyme treatments selected from the group consisting of pectinase, glucoamylase, and lipase may be used to treat the cellulose-containing biomass in step (1) before hydrolysis of cellulase in step (1), or alone during hydrolysis. Although they may be carried out in combination, it is preferred to carry out at least the hydrolysis of the cellulase in step (1).
- the amount of one or more enzymes selected from the group consisting of pectinase, glucoamylase and lipase in the first stage of step (1) or in step (1) but filamentous fungi are used from the viewpoint of reducing enzyme costs. It is preferable that it is 1/10 or less in the weight ratio with respect to origin cellulase.
- the amount of enzyme added here is the total amount of one or more enzymes selected from the group consisting of pectinase, glucoamylase and lipase in the previous stage of step (1) or in step (1).
- the lower limit of the addition amount of the enzyme is not particularly limited as long as the effect of the present invention is obtained, but is 0.001 mg / g biomass in terms of the weight ratio of the enzyme added to the weight of the cellulose-containing biomass.
- the hydrolyzate obtained in step (1) is preferably subjected to solid-liquid separation.
- Solid-liquid separation efficiently separates and recovers sugar solution and filamentous fungus-derived cellulase in step (2) by separating it into a solution component containing sugar (hereinafter also referred to as an aqueous sugar solution) and a saccharification residue that is a solid content.
- a solution component containing sugar hereinafter also referred to as an aqueous sugar solution
- a saccharification residue that is a solid content.
- the purpose is to be able to implement well.
- the method of solid-liquid separation is not particularly limited, and the solid-liquid separation can be performed by centrifugal separation such as a screw decanter, press filtration such as a filter press and a belt press.
- centrifugal separation such as a screw decanter
- press filtration such as a filter press and a belt press
- it is a press filtration such as a filter press or a belt press
- a solution component with less insoluble solids and less turbidity can be obtained compared to centrifugal separation.
- Less turbidity is preferable from the viewpoint of suppressing fouling of the ultrafiltration membrane in the subsequent stage.
- the solid-liquid separation is press filtration such as a filter press or a belt press
- one or more enzymes selected from the group consisting of pectinase, glucoamylase and lipase in the first stage of step (1) or in step (1).
- the effect of shortening the processing time for solid-liquid separation is also brought about by the processing.
- step (2) it is preferable to filter the sugar aqueous solution obtained by solid-liquid separation with a microfiltration membrane before subjecting it to the step (2). Since the solid content that cannot be separated by solid-liquid separation can be removed by filtration through a microfiltration membrane, step (2) can be carried out more efficiently.
- the microfiltration membrane is a membrane having an average pore diameter of 0.01 ⁇ m to 5 mm.
- the material of the microfiltration membrane is not particularly limited as long as it can remove the solid content that could not be separated by the above-mentioned solid-liquid separation, but cellulose, cellulose ester, polysulfone, polyethersulfone, chlorinated polyethylene And organic materials such as polypropylene, polyolefin, polyvinyl alcohol, polymethyl methacrylate, polyvinylidene fluoride and polytetrafluoroethylene, metals such as stainless steel, and inorganic materials such as ceramic.
- the sugar solution obtained in the present invention can be used as a fermentation raw material, and a chemical can be produced by culturing microorganisms.
- chemical products include substances that are mass-produced in the fermentation industry, such as alcohols, organic acids, amino acids, and nucleic acids.
- alcohols such as ethanol, 1,3-propanediol, 1,4-butanediol, glycerol
- organic acids such as acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, itaconic acid, citric acid, inosine, guanosine, etc.
- Nucleosides nucleotides such as inosinic acid and guanylic acid, and amine compounds such as cadaverine. Furthermore, it can be applied to the production of enzymes, antibiotics, recombinant proteins, and the like.
- PE-M and Tween 80 autoclaved at 121 ° C. for 15 minutes were added 0.01% (w / vol), respectively.
- This preculture medium was inoculated with Trichoderma reesei ATCC 66589 at 1 ⁇ 10 5 cells / mL, cultured at 28 ° C. for 72 hours with shaking at 180 rpm, and precultured (shaking apparatus: TAIOTEC BIO-SHAKER BR-40LF).
- 0.1 mL of the enzyme solution was added to 0.9 mL of 100 mM acetate buffer (pH 5.0) containing each substrate at a concentration of 1 mM, and reacted at 30 ° C.
- the reaction time is 60 minutes when the substrate is pNP-Lac, 10 minutes when pNP-Glc and 30 minutes when pNP-Xyl.
- the reaction is stopped by adding 0.1 mL of 2M aqueous sodium carbonate solution.
- the absorbance at 405 nm was measured (ODtest). As a blank, the absorbance at 405 nm was similarly measured for a substrate solution in which a 2M sodium carbonate aqueous solution and an enzyme solution were added in this order (OD blank).
- the amount of enzyme that produces 1 ⁇ mol of 4-nitrophenol per minute in the above reaction system was defined as 1 U, and the activity value (U / mL) was calculated according to the following formula.
- the mmol molecular extinction coefficient of 4-nitrophenol in the above reaction system is 17.2 L / mmol / cm.
- Example 1 As cellulose-containing biomass, (1) corn hull, (2) soybean hull, (3) straw, and (4) bagasse were used. The pretreatment of the cellulose-containing biomass prior to hydrolysis by the filamentous fungus-derived cellulase was performed under different pretreatment conditions for each raw material as described below.
- Corn hull (pretreatment conditions: dilute sulfuric acid treatment) As the corn hull, a dried product made in China was used (purchased from Gordo Co., Ltd.). To 1 kg of corn hull (dry), 2.3 kg of 13 g / L sulfuric acid water was added so that the water content was 70% (30 mg sulfuric acid / g corn hull). The corn skin to which sulfuric acid water was added was hydrothermally treated at 120 ° C. for 30 minutes using an autoclave. After autoclaving, aqueous ammonia was added to the pretreated product to adjust the pH to 5, which was designated as pretreated product 1.
- Soybean hull (pretreatment conditions: dilute sulfuric acid treatment)
- dry To 1 kg of soybean hull (dry), 2.3 kg of 65 g / L sulfuric acid water was added so that the water content was 70% (150 mg sulfuric acid / g soybean hull).
- the soybean hulls to which sulfuric acid water was added were hydrothermally treated at 120 ° C. for 30 minutes using an autoclave. After autoclaving, aqueous ammonia was added to the pretreated product to adjust the pH to 5, which was designated as pretreated product 2.
- Straw (pretreatment conditions: hydrothermal treatment)
- Straw rice straw was dried from Japan (sold separately from farmers).
- the straw was pulverized with a rotary cutter mill RCM-400 (8 mm mesh) manufactured by Nara Machinery Co., Ltd. at a rotation speed of 420 rpm. Thereafter, hydrothermal treatment was performed.
- the device used was a blasting device (reactor 2L size) manufactured by Nippon Electric Heat Co., Ltd.
- the steam generator used a 40 kW electric boiler. Since the processing temperature is uniquely determined when the set processing pressure is set, the reaction conditions were 215 ° C. for 5 minutes, 200 g of crushed straw was added once under this condition, and the reaction was performed 5 times in total.
- the crushed water-containing solid content was added with 2 L of water, stirred, and separated into a hydrothermal treatment liquid and a solid at 5000 rpm using a laboratory centrifuge “HimacCF7D2” manufactured by Hitachi Koki Co., Ltd.
- the water content of the solid was adjusted to 70% and adjusted to pH 5 using aqueous ammonia. This was designated as Pretreatment 3.
- Bagasse (pretreatment conditions: dilute sulfuric acid treatment) Bagasse was made from Taiwan-made dried products (purchased from Taiwan Sugar Sales Co., Ltd.). The bagasse was pulverized with a rotary cutter mill RCM-400 (20 mm mesh) manufactured by Nara Machinery Co., Ltd. at a rotation speed of 420 rpm. To 1 kg of bagasse (dry), 2.3 kg of 13 g / L sulfuric acid water was added so that the water content was 70% (30 mg sulfuric acid / g bagasse). Bagasse to which sulfuric acid water was added was hydrothermally treated using an autoclave at 120 ° C. for 30 minutes. After autoclaving, aqueous ammonia was added to the pretreated product to adjust the pH to 5, which was designated as pretreated product 4.
- filamentous fungus-derived cellulase prepared in Reference Example 1 was added for hydrolysis.
- the amount of filamentous fungus-derived cellulase added is 2 mg / g pre-treated product in pre-treated product 1, 4 mg / g pre-treated product in pre-treated product 2, 8 mg / g pre-treated product in pre-treated product 3 and pre-treated product 4 It was added at 8 mg / g pretreatment and hydrolysis was started. Hydrolysis was carried out at 50 ° C. for 48 hours.
- pectinase (Amano Enzyme Co., Ltd., “Pectinase PL“ Amano ”), glucoamylase (Megazyme Co.,“ AMYLOGLUCOSIDASE ”), lipase (Amano Enzyme Co., Ltd.,“ Lipase AS ”) Amano ”) was added to the combinations shown in Table 1 (the amount added was 0.2 mg / g pre-treated when adding one type.
- the enzyme in the non-permeate obtained after the ultrafiltration treatment was defined as a recovered enzyme, and the activity of the recovered enzyme was measured by the method for measuring the activity of filamentous fungus-derived cellulase in Reference Example 3.
- Tables 2 to 5 show the activity of the recovered enzyme with respect to the filamentous fungus cellulase introduced.
- the method for producing a sugar liquid in the present invention uses cellulose-containing biomass as a raw material, and the obtained sugar liquid can be used as a raw material for fermentation of various chemical products.
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Abstract
Description
[1]セルロース含有バイオマスから糖液を製造する方法であって、
セルロース含有バイオマスを糸状菌由来セルラーゼで加水分解する工程(1)および工程(1)で得られた加水分解物を限外濾過膜に通じて濾過し、非透過液として糸状菌由来セルラーゼを回収し、透過液として糖液を得る工程(2)を含み、
工程(1)の前段または工程(1)中で、ペクチナーゼ、グルコアミラーゼおよびリパーゼからなる群から選ばれる1種以上の酵素でセルロース含有バイオマスを処理する、糖液の製造方法。
[2]前記セルロース含有バイオマスが、穀物皮類バイオマス、わらおよびバガスからなる群から選ばれる1種以上である、[1]に記載の糖液の製造方法。
[3]前記穀物皮類バイオマスが、コーン外皮、大豆外皮および小麦外皮からなる群から選ばれる1種以上である、[2]に記載の糖液の製造方法。
[4]前記ペクチナーゼ、グルコアミラーゼおよびリパーゼからなる群から選ばれる1種以上の酵素の重量が、糸状菌由来セルラーゼの重量に対して1/10以下である、[1]から[3]のいずれかに記載の糖液の製造方法。
[5]前記工程(1)の前段または前記工程(1)中で、ペクチナーゼ、グルコアミラーゼおよびリパーゼの3種の酵素でセルロース含有バイオマスを処理する、[1]から[4]のいずれかに記載の糖液の製造方法。
[6]前記工程(2)が、工程(1)で得られた加水分解物を固液分離して得られた溶液成分を限外ろ過膜に通じて濾過する工程である、[1]から[5]のいずれかに記載の糖液の製造方法。
[7]前記固液分離がプレス濾過である、[6]に記載の糖液の製造方法。
[工程(1)]
工程(1)は、セルロース含有バイオマスを糸状菌由来セルラーゼで加水分解する工程である。
工程(2)では、前記工程(1)で得られた加水分解物を限外濾過膜に通じて濾過し、非透過液として糸状菌由来セルラーゼを回収し、透過液として糖液を得る。非透過液として回収した酵素は、工程(1)に再利用することも可能であり、工程(1)での酵素の使用量を低減することが可能となる。
本発明は、工程(1)の前段または工程(1)中で、ペクチナーゼ、グルコアミラーゼおよびリパーゼからなる群から選ばれる1種以上の酵素でセルロース含有バイオマスを処理することを特徴とする。工程(1)の前段または工程(1)中で、ペクチナーゼ、グルコアミラーゼおよびリパーゼからなる群から選ばれる1種以上の酵素でセルロース含有バイオマスを処理することにより、処理しない場合と比較して工程(2)で回収されうる糸状菌由来セルラーゼ量が増大するといった効果が得られ、特に、糸状菌由来セルラーゼに含まれる酵素成分のうち、セルロース含有バイオマスを加水分解するうえで主要な酵素成分であるセロビオハイドロラーゼの回収量が顕著に増大するという効果が得られる。
その他、工程(1)で得られた加水分解物を固液分離することが好ましい。固液分離は、糖を含む溶液成分(以下、糖水溶液ともいう。)と、固形分である糖化残さに分離することで工程(2)での糖液と糸状菌由来セルラーゼの分離回収を効率よく実施できるようにすることを目的とする。
糸状菌由来セルラーゼ(培養液)は、次の方法で調製した。
コーンスティップリカー5%(w/vol)、グルコース2%(w/vol)、酒石酸アンモニウム0.37%(w/vol)、硫酸アンモニウム0.14(w/vol)、リン酸二水素カリウム0.2%(w/vol)、塩化カルシウム二水和物0.03%(w/vol)、硫酸マグネシウム七水和物0.03%(w/vol)、塩化亜鉛0.02%(w/vol)、塩化鉄(III)六水和物0.01%(w/vol)、硫酸銅(II)五水和物0.004%(w/vol)、塩化マンガン四水和物0.0008%(w/vol)、ホウ酸0.0006%(w/vol)、および七モリブデン酸六アンモニウム四水和物0.0026%(w/vol)となるように蒸留水に添加し、100mLを500mLバッフル付き三角フラスコに張り込み、121℃の温度で15分間オートクレーブ滅菌した。放冷後、これとは別に、それぞれ121℃の温度で15分間オートクレーブ滅菌したPE-MとTween80を、それぞれ0.01%(w/vol)添加した。この前培養培地に、トリコデルマ・リーセイATCC66589を1×105個/mLになるように植菌し、28℃の温度で72時間、180rpmで振とう培養し、前培養とした(振とう装置:TAITEC社製 BIO-SHAKER BR-40LF)。
コーンスティップリカー5%(w/vol)、グルコース2%(w/vol)、セルロース(アビセル)10%(w/vol)、酒石酸アンモニウム0.37%(w/vol)、硫酸アンモニウム0.14%(w/vol)、リン酸二水素カリウム0.2%(w/vol)、塩化カルシウム二水和物0.03%(w/vol)、硫酸マグネシウム七水和物0.03%(w/vol)、塩化亜鉛0.02%(w/vol)、塩化鉄(III)六水和物0.01%(w/vol)、硫酸銅(II)五水和物0.004%(w/vol)、塩化マンガン四水和物0.0008%(w/vol)、ホウ酸0.0006%(w/vol)、および七モリブデン酸六アンモニウム四水和物0.0026%(w/vol)となるように蒸留水に添加し、2.5Lを5L容撹拌ジャー(ABLE社製、DPC-2A)容器に張り込み、121℃の温度で15分間オートクレーブ滅菌した。放冷後、これとは別に、それぞれ121℃の温度で15分間オートクレーブ滅菌したPE-MとTween80を、それぞれ0.1%添加し、あらかじめ前記の方法で液体培地で前培養したトリコデルマ・リーセイATCC66589を250mL接種した。その後、28℃の温度で87時間、300rpm、通気量1vvmの条件で振とう培養を行い、遠心分離後、上清を膜ろ過(ミリポア社製のステリカップ-GV、材質:PVDF)した。この前述条件で調整した培養液を糸状菌由来セルラーゼとして、以下の実施例に使用した。
糖液に含まれるグルコースおよびキシロース濃度は、下記に示すHPLC条件で、標品との比較により定量した。
カラム:Luna NH2(Phenomenex社製)
移動相:ミリQ:アセトニトリル=25:75(流速0.6mL/分)
反応液:なし
検出方法:RI(示差屈折率)
温度:30℃。
セルラーゼ活性として、セルロースの分解に関与するセロビオハイドロラーゼ、エンドグルカナーゼの活性を(1)4-ニトロフェニル-β-D-ラクトピラノシド(pNP-Lac)分解活性より、βグルコシダーゼの活性を(2)4-ニトロフェニル-β-D-グルコピラノシド(pNP-Glc)の分解活性より、また、ヘミセルロースの主成分であるキシランの分解に関与するエンドキシラナーゼ、キシロシダーゼの活性を(3)4-ニトロフェニル-β-D-キシロピラノシド(pNP-Xyl)の分解活性より求めた。各分解活性の測定方法を以下に示す。なお、上記(1)~(3)の基質をまとめてpNP-糖という。
pNP-Lac分解活性(U/mL)={(ODtest-ODblank)×1.1(mL)×酵素希釈倍率}/{17.2×60(分間)×0.1(mL)}
pNP-Glc分解活性(U/mL)={(ODtest-ODblank)×1.1(mL)×酵素希釈倍率}/{17.2×10(分間)×0.1(mL)}
pNP-Xyl分解活性(U/mL)={(ODtest-ODblank)×1.1(mL)×酵素希釈倍率}/{17.2×60(分間)×0.1(mL)}。
セルロース含有バイオマスとして、(1)コーン外皮、(2)大豆外皮、(3)わら、(4)バガスを使用した。糸状菌由来セルラーゼによる加水分解に先立つセルロース含有バイオマスの前処理は、下記のとおり原料毎に異なる前処理条件にて処理を行った。
コーン外皮は、中国産の乾燥品を使用した(株式会社ゴードーより購入)。コーン外皮(乾燥)1kgに対して、含水率70%となるように13g/L硫酸水2.3kgを添加した(30mg硫酸/gコーン外皮)。硫酸水を添加したコーン外皮は、120℃で30分オートクレーブを使用して水熱処理した。オートクレーブ後、前処理物にアンモニア水を添加し、pH5に調製し、これを前処理物1とした。
大豆外皮は、中国産の乾燥品を使用した(株式会社ゴードーより購入)。大豆外皮(乾燥)1kgに対して、含水率70%となるように65g/L硫酸水2.3kgを添加した(150mg硫酸/g大豆外皮)。硫酸水を添加した大豆外皮は、120℃で30分オートクレーブを使用して水熱処理した。オートクレーブ後、前処理物にアンモニア水を添加してpH5に調製し、これを前処理物2とした。
わら(稲わら)は、日本産の乾燥品を使用した(農家より分譲)。わらを奈良機械製作所製のロータリーカッターミル・RCM-400(8mmメッシュ)にて回転速度420回転/分で粉砕した。その後、水熱処理を行った。装置は、日本電熱株式会社製の爆砕装置(反応器2Lサイズ)を使用した。蒸気発生装置は40kWの電気ボイラを使用した。設定した処理圧力を設定すると一義的に処理温度も決定するため、反応条件は、215℃で5分処理を行い、本条件で1回200gの粉砕したわらを投入し、合計5回実施した。爆砕処理した含水した固形分を2Lの水を加えて攪拌し、日立工機株式会社製のラボ用遠心分離機“HimacCF7D2”を用いて5000rpmで水熱処理液と固形物に分離した。固形物の含水率は70%になるよう調製しアンモニア水を使用してpH5に調製した。これを前処理物3とした。
バガスは、台湾製の乾燥品を使用した(台糖農産販売株式会社より購入)。バガスを奈良機械製作所製のロータリーカッターミル・RCM-400(20mmメッシュ)にて回転速度420回転/分で粉砕した。バガス(乾燥)1kgに対して、含水率70%となるように13g/L硫酸水2.3kgを添加した(30mg硫酸/gバガス)。硫酸水を添加したバガスは、120℃で30分オートクレーブを使用して水熱処理した。オートクレーブ後、前処理物にアンモニア水を添加し、pH5に調製し、これを前処理物4とした。
Claims (7)
- セルロース含有バイオマスから糖液を製造する方法であって、
セルロース含有バイオマスを糸状菌由来セルラーゼで加水分解する工程(1)および工程(1)で得られた加水分解物を限外濾過膜に通じて濾過し、非透過液として糸状菌由来セルラーゼを回収し、透過液として糖液を得る工程(2)を含み、
工程(1)の前段または工程(1)中で、ペクチナーゼ、グルコアミラーゼおよびリパーゼからなる群から選ばれる1種以上の酵素でセルロース含有バイオマスを処理する、糖液の製造方法。 - 前記セルロース含有バイオマスが、穀物皮類バイオマス、わらおよびバガスからなる群から選ばれる1種以上である、請求項1に記載の糖液の製造方法。
- 前記穀物皮類バイオマスが、コーン外皮、大豆外皮および小麦外皮からなる群から選ばれる1種以上である、請求項2に記載の糖液の製造方法。
- 前記ペクチナーゼ、グルコアミラーゼおよびリパーゼからなる群から選ばれる1種以上の酵素の重量が、糸状菌由来セルラーゼの重量に対して1/10以下である、請求項1から3のいずれかに記載の糖液の製造方法。
- 前記工程(1)の前段または前記工程(1)中で、ペクチナーゼ、グルコアミラーゼおよびリパーゼの3種の酵素でセルロース含有バイオマスを処理する、請求項1から4のいずれかに記載の糖液の製造方法。
- 前記工程(2)が、工程(1)で得られた加水分解物を固液分離して得られた溶液成分を限外ろ過膜に通じて濾過する工程である、請求項1から5のいずれかに記載の糖液の製造方法。
- 前記固液分離がプレス濾過である、請求項6に記載の糖液の製造方法。
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JPWO2017170918A1 (ja) * | 2016-03-31 | 2019-02-07 | 東レ株式会社 | 変異型bxl1遺伝子を有するトリコデルマ属真菌及びそれを使用したキシロオリゴ糖とグルコースの製造方法 |
US10590497B2 (en) | 2016-03-31 | 2020-03-17 | Toray Industries, Inc. | Trichoderma fungus having mutant-type BXL1 gene and method of producing xylooligosaccharide and glucose by using same |
JP7007645B2 (ja) | 2016-03-31 | 2022-02-10 | 東レ株式会社 | 変異型bxl1遺伝子を有するトリコデルマ属真菌及びそれを使用したキシロオリゴ糖とグルコースの製造方法 |
US11453899B2 (en) | 2016-03-31 | 2022-09-27 | Toray Industries, Inc. | Method of producing protein |
WO2018159573A1 (ja) * | 2017-02-28 | 2018-09-07 | 東レ株式会社 | 糖化酵素の製造方法およびオリゴ糖の製造方法 |
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MY176473A (en) | 2020-08-11 |
EP3088530A1 (en) | 2016-11-02 |
EP3088530B1 (en) | 2020-04-29 |
EP3088530A4 (en) | 2017-08-09 |
CN105745331B (zh) | 2019-12-20 |
BR112016014736A2 (pt) | 2020-09-08 |
JPWO2015099109A1 (ja) | 2017-03-23 |
CN105745331A (zh) | 2016-07-06 |
US11155848B2 (en) | 2021-10-26 |
US20160326559A1 (en) | 2016-11-10 |
CA2935043A1 (en) | 2015-07-02 |
JP6508039B2 (ja) | 2019-05-08 |
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