JP2014064563A - Method for producing sugar liquid - Google Patents

Abstract

PROBLEM TO BE SOLVED: To heighten reusability of cellulase derived from filamentous fungus and to reduce a use amount thereof, in a hydrolysis reaction of a cellulose-containing biomass using the cellulase derived from filamentous fungus.SOLUTION: A method for producing sugar liquid from a cellulose-containing biomass comprises: a step (1) of hydrolyzing the cellulose-containing biomass by using cellulase derived from filamentous fungus under a condition that 0.1 to 50 wt.% of a fermentation residue, with respect to solid weight of the cellulose-containing biomass is included; and a step (2) of filtering a solution component obtained by solid-liquid separation of hydrolyzate obtained by the step (1), and collecting the cellulase derived from filamentous fungus as the non-permeate to obtain sugar liquid as the permeate.

Description

  The present invention relates to a method for producing a sugar solution from cellulose-containing biomass.

  In recent years, a method for hydrolyzing cellulose-containing biomass using an enzyme, which has a particularly low energy consumption and an environmental load and has a high sugar yield, has been widely studied. However, the biggest drawback of the method using an enzyme is that the enzyme cost is high. In order to solve these technical problems, a method for recovering and reusing the enzyme used for hydrolysis has been proposed, but the enzyme strongly adsorbs to the hydrolysis residue generated when hydrolyzing cellulose-containing biomass. The problem is low reusability.

  As a method for desorbing the enzyme adsorbed on the hydrolysis residue and increasing the recovery rate of the enzyme, a method of washing the hydrolysis residue with an alkaline aqueous solution having a pH of about 8 (Non-patent Document 1), a nonionic substance on a hydrolyzate of cellulose-containing biomass A method of adding a functional surfactant (Patent Document 1) is known. On the other hand, as a method for reducing the adsorption of the enzyme to the hydrolysis residue, a method of adjusting the electrical conductivity of the reaction solution to 5 to 25 mS / cm by adding water-soluble salts during hydrolysis of the cellulose-containing biomass (patent) Document 2), a method of adding 1 to 10% by weight of calcium carbonate particles to the solid weight of cellulose-containing biomass (Patent Document 3), and the like are known.

JP-A-63-87994 Japanese Patent No. 4947223 JP 2012-1000061 A

D. E. Otter et al., “Elution of Trichoderma reesei Cellulose from Cellulose by pH Adjustment with Sodium Hydroxide” Biotechnology Letters (1984) Vol. 6, No. 6, 369-374

  As described above, various attempts have been made to reduce the amount of enzyme used by recovering and reusing the enzyme used for hydrolysis of cellulose-containing biomass. However, the enzyme is strongly adsorbed to the hydrolysis residue. The recovery rate is low and the problem has not been solved.

  Therefore, an object of the present invention is to provide a sugar solution that is produced by a method with high enzyme reusability and that can be easily used as a fermentation raw material for chemical production using microorganisms.

  As a result of intensive studies by the present inventors for solving the above-mentioned problems, the hydrolysis of cellulose-containing biomass by filamentous fungus-derived cellulase is carried out in the presence of the fermentation residue of the starch raw material. The present inventors have found that it can be recovered with high efficiency and have completed the present invention.

That is, the present invention has the following configurations [1] to [7].
[1] A method for producing a sugar liquid from cellulose-containing biomass, comprising the following steps (1) and (2).
Step (1): The cellulose-containing biomass is hydrolyzed by the filamentous fungus-derived cellulase under the condition containing the fermentation residue of the starch raw material in the range of 0.1 to 50% by weight with respect to the solid weight of the cellulose-containing biomass. Process,
Step (2): The solution component obtained by solid-liquid separation of the hydrolyzate in Step (1) is filtered through an ultrafiltration membrane, and the filamentous fungus-derived cellulase is recovered as a non-permeate and used as a permeate A step of obtaining a sugar solution.
[2] The filamentous fungus-derived cellulase in the step (1) contains one or more enzyme components selected from the group consisting of cellobiohydrase, endoglucanase, β-glucosidase, xylanase and β-xylosidase, The method for producing a sugar liquid according to [1].
[3] The method for producing a sugar liquid according to [1] or [2], wherein the fermentation residue of the starch raw material in step (1) is a distillation residue after ethanol fermentation.
[4] The method for producing a sugar liquid according to any one of [1] to [3], wherein in the step (1), the diluted sulfuric acid-treated product or hydrothermally-treated product of cellulose-containing biomass is hydrolyzed.
[5] In the step (1), a water-soluble inorganic salt and / or a polar organic solvent is contained in a concentration range of 0.1 to 5% by weight with respect to the total hydrolysis reaction amount. 4] The method for producing a sugar liquid according to any one of the above.
[6] The method for producing a sugar liquid according to any one of [1] to [5], further comprising the following steps (3) and (4):
Step (3): A step of washing the hydrolysis residue obtained by solid-liquid separation in Step (2) with washing water containing one or more selected from the group consisting of a water-soluble inorganic salt, an alkali, and a polar organic solvent,
Step (4): A step of solid-liquid separation of the washed product of step (3) to separate a hydrolysis residue and a washing solution, and recovering a filamentous fungus-derived cellulase component contained in the washing solution.
[7] A microorganism having the ability to produce a chemical using the sugar solution obtained by the method for producing a sugar solution according to any one of [1] to [6] as a fermentation raw material, is fermented and cultured. , Chemical manufacturing method.

  ADVANTAGE OF THE INVENTION According to this invention, the adsorption | suction of the filamentous fungus origin cellulase to the hydrolysis residue of a cellulose containing biomass can be suppressed, and especially the enzyme component which is concerned in decomposition | disassembly of crystalline cellulose and cellobiose is collect | recovered and / or reused with high efficiency. can do. As a result, the cost of the enzyme can be kept low.

FIG. 1 is a schematic view showing an embodiment of the method for producing a sugar liquid of the present invention. FIG. 2 is a schematic diagram showing an embodiment of washing a hydrolysis residue in the sugar liquid production method of the present invention.

  Hereinafter, the form for implementing this invention is demonstrated for every process.

  First, in the present invention, the cellulose-containing biomass is hydrolyzed by the filamentous fungus-derived cellulase under the condition containing the fermentation residue of the starch raw material in the range of 0.1 to 50% by weight with respect to the solid weight of the cellulose-containing biomass. (Step (1)).

  Cellulose-containing biomass includes herbaceous biomass such as bagasse, switchgrass, napiergrass, eliansus, corn stover, beet pulp, cottonseed husk, palm husk, rice straw, straw, bamboo, straw, etc., or birch, beech, etc. Woody biomass such as wood and waste building materials. Since cellulose-containing biomass contains lignin, which is an aromatic polymer, in addition to cellulose and hemicellulose composed of sugar, hydrolysis efficiency by enzymes can be improved by pretreatment. Examples of the pretreatment method for cellulose-containing biomass include acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, alkali treatment, caustic soda treatment, ammonia treatment, hydrothermal treatment, subcritical water treatment, pulverization treatment, and steaming treatment. In the method for producing a sugar solution, the reusability of the enzyme is highest when a dilute sulfuric acid treatment product or a hydrothermal treatment product is used, and these pretreatment products can be preferably used.

  In the present invention, a cellulase derived from a filamentous fungus is used for hydrolysis of cellulose-containing biomass. The filamentous fungi include Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Humicola, and Humicola. Examples thereof include microorganisms such as the genus Irpex (Irpex), the genus Mucor (Mucor), and the genus Talaromyces (Talaromyces). In addition, a cellulase derived from a mutant strain in which cellulase productivity has been improved by subjecting these microorganisms to mutation treatment or irradiation with ultraviolet rays or the like may be used.

  Among the filamentous fungi, Trichoderma spp. Can be preferably used in the present invention because an enzyme component having a high specific activity in the hydrolysis of cellulose is produced in a large amount in the culture solution. Specific examples of cellulases derived from the genus Trichoderma include Trichoderma reesei QM9414 (Trichoderma reesei QM9414), Trichoderma reesei QM9123 (Trichoderma reesei QM9123), Trichoderma reesei Rutc-30 R3 (Trichoderma reesei PC3-7), Trichoderma reesei CL-847 (Trichoderma reesei CL-847), Trichoderma reesei MCG77 (Trichoderma reesei MCG77), Trichoderma reesei MCG80 (TriCode 80) Although cellulase derived from Ruma viride QM9123 (Trichoderma violet QM9123) is mentioned, Trichoderma reesei cellulase is more preferable.

  The filamentous fungus-derived cellulase is an enzyme composition having an activity of hydrolyzing cellulose and / or hemicellulose to produce monosaccharides such as glucose and xylose. As an enzyme component, cellobiohydrase, endoglucanase, β-glucosidase, It is preferable to include one or more selected from the group consisting of xylanase and β-xylosidase. For example, as an enzyme component of cellulase derived from Trichoderma reesei, cellobiohydrase I, cellobiohydrase II, endoglucanase I, endoglucanase III, β-glucosidase, xylanase, β-xylosidase, etc. are exemplified. Since the hydrolysis of cellulose and / or hemicellulose can be carried out efficiently by the concerted effect or the complementary effect of the enzyme component, it is preferably used in the present invention.

  Cellobiohydrase is a general term for enzymes that release cellobiose by hydrolysis of cellulose chains, and an enzyme group belonging to cellobiohydrase is described as EC number: EC 3.2.1.91. Cellobiohydrase I starts the hydrolysis reaction from the reducing end side of the cellulose chain, and cellobiohydrase II starts the hydrolysis reaction from the non-reducing end side.

  Endoglucanase is a general term for enzymes characterized by hydrolysis from the central part of the cellulose chain, and an enzyme group belonging to endoglucanase is described as EC number: EC3.2.1.4. Endoglucanase I is the most expressed endoglucanase produced by Trichoderma reesei cellulase and has a wide substrate specificity. Endoglucanase III does not have a cellulose binding module (CBM) and has a feature of low molecular weight.

  β-glucosidase is a general term for enzymes characterized by acting on cellooligosaccharide or cellobiose, and an enzyme group belonging to β-glucosidase is described as EC number: EC 3.2.1.21.

  Xylanase is a general term for enzymes characterized by acting on hemicellulose or in particular xylan, and an enzyme group belonging to xylanase is described as EC number: EC3.2.1.8.

  β-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.

  These cellulase components are separated by known methods such as gel filtration, ion exchange, and two-dimensional electrophoresis, and amino acid sequences (N-terminal analysis, C-terminal analysis, mass spectrometry) of the separated components are identified and identified by comparison with a database. can do.

  The enzymatic activity of cellulase derived from filamentous fungi can be evaluated by polysaccharide hydrolytic activity such as crystalline cellulose degrading activity, carboxymethyl cellulose (CMC) degrading activity, cellobiose degrading activity, xylan degrading activity, mannan degrading activity and the like. The main enzyme exhibiting crystalline cellulose-degrading activity is cellobiohydrase, which has the characteristic of hydrolyzing from the terminal portion of cellulose. The main enzyme exhibiting cellobiose degradation activity is β-glucosidase. The main enzymes involved in CMC degradation activity are cellobiohydrase and endoglucanase. The main enzymes showing xylan degradation activity are xylanase and β-xylosidase. Here, the term “principal” is an expression from what is known to be most involved in degradation, and means that other enzyme components are also involved in the degradation.

  Since the filamentous fungus produces cellulase in the culture solution, the culture solution may be used as it is as a crude enzyme agent, or the enzyme group is purified by a known method and formulated into a filamentous fungus-derived cellulase mixture. May be used as When a filamentous fungus-derived cellulase is purified and used as a preparation, a substance added with a substance other than an enzyme such as a protease inhibitor, a dispersant, a dissolution accelerator, or a stabilizer may be used as a cellulase preparation. . In the present invention, among these, a crude enzyme product is preferably used. 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 a medium to which cellulose has been added in order to promote cellulase production can be generally used. As the crude enzyme product, the culture supernatant is preferably used as it is, or the culture supernatant from which Trichoderma cells are removed.

  The weight ratio of each enzyme component in the crude enzyme product is not particularly limited. For example, the culture solution derived from Trichoderma reesei contains 50 to 95% by weight of cellobiohydrase, and the rest The endoglucanase, β-glucosidase and the like are included in the components. In addition, Trichoderma microorganisms produce powerful cellulase components in the culture solution, while β-glucosidase retains most of it in the cell or on the cell surface, so β-glucosidase activity in the culture solution Is low. Therefore, a heterogeneous or homologous β-glucosidase may be further added to the crude enzyme product. As the heterogeneous β-glucosidase, β-glucosidase derived from the genus Aspergillus can be preferably used. Examples of β-glucosidase derived from the genus Aspergillus include Novozyme 188 commercially available from Novozyme. Alternatively, a culture solution having improved β-glucosidase activity may be used by introducing a gene into a Trichoderma microorganism, culturing the Trichoderma microorganism that has been genetically modified to be produced in the culture solution.

  The temperature of the hydrolysis reaction is preferably in the range of 40 to 60 ° C, and more preferably in the range of 45 to 55 ° C, particularly when Trichoderma-derived cellulase is used. The time for the hydrolysis reaction is preferably in the range of 2 hours to 200 hours. If it is less than 2 hours, sufficient sugar production may not be obtained. On the other hand, if it exceeds 200 hours, the deactivation of the enzyme proceeds, and the reusability of the recovered enzyme may be adversely affected. The pH of the hydrolysis reaction is preferably in the range of 4.0 to 6.0. When Trichoderma-derived cellulase is used as the filamentous fungus-derived cellulase, the optimum pH for the reaction is 5.0. Furthermore, since a change in pH occurs during the hydrolysis, it is preferable to add a buffer solution to the reaction solution or to maintain a constant pH using an acid or alkali.

  The concentration of the cellulose-containing biomass in the hydrolysis reaction is preferably in the range of 1 to 30% by weight. When the concentration of cellulose-containing biomass is low, the sugar concentration produced by hydrolysis is low, and it may be difficult to use as a fermentation raw material. On the other hand, if the concentration is too high, handling becomes difficult.

  In the method for producing a sugar solution of the present invention, the hydrolysis reaction of cellulose-containing biomass by filamentous fungus-derived cellulase is carried out in the presence of a fermentation residue of a starch raw material. Fermentation residue of starch raw material contains a component that strongly adsorbs to hydrolysis residue of cellulose-containing biomass, so that adsorption of cellulase component to hydrolysis residue can be suppressed, and cellulase component is recovered with high efficiency can do.

  The starch raw material is not particularly limited as long as it contains starch abundantly, and examples thereof include corn, rice, wheat, barley, buckwheat, potato, sweet potato, sorghum, and cassava. By fermenting microorganisms using the starch raw material as a nutrient source, a fermented product of the starch raw material can be obtained. Although the microorganisms used for fermentation are not specifically limited, Lactic acid bacteria, yeast, etc. are mentioned. When it is difficult for microorganisms used for fermentation to use starch as a nutrient source, a product obtained by saccharifying starch by acting amylase or heating under acidic conditions can be used.

  And the fermentation residue of a starch raw material refers to the liquid containing the solid substance obtained by solid-liquid-separating fermented material, or the solid substance obtained by distilling fermented material. A specific example of the former is sake lees. The latter is generically referred to as distillation residue, and specific examples include shochu, whiskey cake and the like.

  In recent years, bioethanol production using starch raw materials represented by corn has been actively carried out from the viewpoint of using renewable energy. Therefore, these fermentation residues can be preferably used in the present invention because they are supplied in a large amount and can be obtained at low cost. In addition to distillation residues (hole distilage), solids (wet cakes) obtained by solid-liquid separation of these products, soluble material-added dry distilled cereal residues (DDGS) obtained by drying these, and dry distilled cereal residues ( DDG) or the like can be used as a fermentation residue.

  Constituent components of the fermentation residue of the starch raw material include proteins, fats and dietary fibers derived from the raw material, and yeast and lactic acid bacteria used for fermentation. For example, in the case of the above-mentioned corn DDG, although there are variations depending on the production method, in general, components other than starch contained in the raw material corn are concentrated about 3 times, and further about 10% of cells are included. .

  The fermentation residue amount (dry weight) of the starch raw material in the hydrolysis reaction of the cellulose-containing biomass is 0.1 to 50% by weight, preferably 1 to 20% by weight, based on the solid weight of the cellulose-containing biomass to be subjected to hydrolysis. More preferably, it is added in the range of 5 to 10% by weight. If the amount of fermentation residue of the starch raw material is less than 1% by weight, the effect of preventing adsorption of cellulase to the hydrolysis residue may not be sufficiently obtained. On the other hand, if the amount of fermentation residue exceeds 20% by weight, it is economically disadvantageous, and since the fermentation residue is a solid, the hydrolysis efficiency of cellulose-containing biomass may deteriorate.

  The present invention is characterized in that the cellulose-containing biomass is hydrolyzed in the presence of the fermentation residue of the starch raw material. In order to recover and / or reuse more cellulase components, a water-soluble inorganic substance is further added during the hydrolysis reaction. Salts and / or polar organic solvents may be added.

  The water-soluble inorganic salt is not particularly limited, and examples thereof include one or more selected from sodium salt, potassium salt, magnesium salt, sulfate, ammonium salt, hydrochloride, phosphate, and nitrate. More preferable inorganic salts include sodium chloride, sodium sulfate, sodium hydrogen sulfate, sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium sulfate, potassium chloride, ammonium chloride, magnesium chloride, magnesium sulfate and the like having high water solubility. Of these, sodium chloride, potassium chloride, magnesium chloride, and ammonium sulfate are most preferred. By adding such inorganic salts, a large amount of cellulase components particularly related to cellobiose decomposition activity can be recovered.

  The polar organic solvent is not particularly limited, but indicates a solvent having a solubility of 10% by weight or more in water at 20 ° C., and includes methanol, ethanol, 1-propanol, isopropanol, dimethyl sulfoxide, N, N-dimethylformamide. , Acetone, acetonitrile, ethylene glycol, glycerol and the like. Among these, ethanol, isopropanol, ethylene glycol, and glycerol that are effective for recovering cellulase components involved in crystalline cellulose decomposition activity are preferable.

  The addition concentration of the water-soluble inorganic salt and / or polar organic solvent is preferably in the range of 0.1 to 5% by weight and more preferably in the range of 0.1 to 3.5% by weight with respect to the total hydrolysis amount. If the concentration of the water-soluble inorganic salt and / or polar organic solvent is too low, the recovery efficiency of cellulase may be low. On the other hand, if the concentration is too high, the deactivation of cellulase is promoted and hydrolysis of cellulose-containing biomass is promoted. The efficiency may be reduced, the cellulase recovery efficiency may be lowered, and it is economically disadvantageous.

  Next, in this invention, after hydrolyzing a cellulose containing biomass, a hydrolyzate is solid-liquid separated. The solution component obtained by solid-liquid separation contains a cellulase component and a sugar component derived from a filamentous fungus, and the cellulase component and the sugar component derived from a filamentous fungus are separated by filtration using an ultrafiltration membrane (step (2)). .

  In the separation of filamentous fungus-derived cellulase and sugar components using an ultrafiltration membrane, the molecular weight cut-off can pass through monosaccharides such as glucose (molecular weight 180) and xylose (molecular weight 150), There is no limitation as long as it can be prevented. Specifically, the molecular weight cut off may be in the range of 500 to 50,000, and from the viewpoint of separating a contaminant having an inhibitory action on the enzyme reaction from the enzyme, more preferably the molecular weight cut off is 5,000 to 50, 000, and more preferably a molecular weight cut-off 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. Sulfone, polyolefin, polyvinyl alcohol, polymethyl methacrylate, polytetrafluoroethylene, etc. can be used, but since regenerated cellulose, cellulose, and cellulose ester are decomposed by cellulase, synthetic polymers such as PES and PVDF are used as materials. It is preferable to use an ultrafiltration membrane as described above.

  Examples of the filtration method of the ultrafiltration membrane include dead-end filtration and crossflow filtration. From the viewpoint of suppressing membrane fouling, crossflow filtration is preferable. Moreover, as a membrane form of the ultrafiltration membrane to be used, those having an appropriate form such as a flat membrane type, a spiral type, a tubular type, and a hollow fiber type can be used. Specifically, DESAL G-5 type, G-10 type, G-20 type, G-50 type, PW type, HWSUF type, KOCH HFM-180, HFM-183, HFM-251, HFM -300, HFK-131, HFK-328, MPT-U20, MPS-U20P, MPS-U20S, Sender 1, SPE3, SPE5, SPE10, SPE30, SPV5, SPV50, SOW30, Microza made by Asahi Kasei Corporation Examples thereof include those corresponding to a molecular weight cutoff of 3,000 to 10,000 in the registered trademark UF series, NTR7410 and NTR7450 manufactured by Nitto Denko Corporation.

  In addition, in order to recover and / or reuse more cellulase components, hydrolysis is performed by washing the resulting hydrolysis residue after solid-liquid separation of the cellulose-containing biomass hydrolyzate in step (2). The cellulase component adsorbed on the residue can also be recovered (step (3)).

  Washing the hydrolysis residue refers to immersing the hydrolysis residue in arbitrary washing water. The washing water for the hydrolysis residue is not particularly limited, but water containing one or more selected from the group consisting of water-soluble inorganic salts, alkalis, and polar organic solvents can be preferably used.

  The type of water-soluble inorganic salt added to the washing water of the hydrolysis residue is not particularly limited, but one type selected from sodium salt, potassium salt, magnesium salt, sulfate salt, ammonium salt, hydrochloride salt, phosphate salt and nitrate salt Or 2 or more types can be illustrated. From the viewpoint of high water solubility, more preferable inorganic salts are sodium chloride, sodium sulfate, sodium hydrogen sulfate, sodium dihydrogen phosphate, disodium hydrogen phosphate, ammonium sulfate, potassium chloride, ammonium chloride, magnesium chloride, magnesium sulfate. is there. More preferred are sodium chloride, potassium chloride, magnesium chloride, and ammonium sulfate. By adding these water-soluble inorganic salts to the washing water of the hydrolysis residue, particularly cellulose components involved in cellobiose decomposition activity and xylan decomposition activity are increased. It can be recovered.

  The addition concentration of the water-soluble inorganic salt is preferably in the range of 0.05 to 5% by weight. If the concentration of the water-soluble inorganic salt is too low, the cellulase recovery efficiency may be low. On the other hand, if the concentration of the water-soluble inorganic salt is too high, the deactivation of cellulase may be promoted, which is disadvantageous economically.

  The alkali added to the washing water of the hydrolysis residue is not particularly limited as long as it is a substance that exhibits a pH higher than 7 when dissolved in water, but alkali metal or alkaline earth metal hydroxide, ammonia, amine, Examples thereof include one or more selected from alkali metal carbonates and alkali metal phosphates. Sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, sodium carbonate, calcium carbonate, trisodium phosphate, etc. are used as more preferable alkalis from the viewpoint that they are well soluble in water and can be adjusted to a desired pH with a small amount. be able to. More preferably, sodium hydroxide, potassium hydroxide, calcium hydroxide or the like whose pH can be adjusted with a small amount can be used. By using washing water containing these alkalis, it is possible to recover a large amount of cellulase components involved in the crystalline cellulose decomposing activity and cellobiose decomposing activity.

  The amount of alkali added is preferably in the range where the pH of the washing water is 7.5 to 10.0. When the pH of the washing water is lower than 7.5, the cellulase component recovery efficiency may be low. On the other hand, when the pH of the washing water is higher than 10.0, the deactivation of the cellulase component is promoted and the recovery efficiency may be lowered.

  The polar organic solvent to be added to the washing water of the hydrolysis residue is not particularly limited, but refers to a solvent having a solubility of 10% by weight or more with respect to water under the condition of 20 ° C., methanol, ethanol, 1-propanol, isopropanol, Examples include dimethyl sulfoxide, N, N-dimethylformamide, acetone, acetonitrile, ethylene glycol, glycerol and the like. Among these, ethanol, isopropanol, ethylene glycol, and glycerol that are effective for recovering components involved in the crystalline cellulose decomposition activity are preferable.

  The addition concentration of the polar organic solvent is preferably in the range of 0.05 to 5% by weight. If the concentration of the polar organic solvent is too low, cellulase recovery efficiency may be low. On the other hand, if the concentration of the polar organic solvent is too high, the deactivation of cellulase may be promoted, which is disadvantageous economically.

  The hydrolysis residue is preferably washed within a range of 25 to 50 ° C. If the temperature is too low, the cellulase recovery efficiency may not be sufficient. On the other hand, if the temperature is too high, the cellulase component may be deactivated and the recovery efficiency may decrease.

  As described above, since the water-soluble inorganic salt, alkali, and polar organic solvent are effective in recovering different cellulase components, washing water containing two or more of these may be used, or washing water having different compositions may be used. The hydrolysis residue may be washed several times independently. Recovery efficiency can be increased by using a cleaning solution suitable for recovery of any cellulase component.

  The washing product of the hydrolysis residue is separated into the hydrolysis residue and the washing solution by solid-liquid separation, and the cellulase component can be recovered and / or reused by filtration using an ultrafiltration membrane as in the step (2) ( Step (4)).

  In addition, the sugar solution obtained by the present invention may be subjected to a concentration treatment for further increasing the sugar concentration. Examples of the concentration treatment include evaporative concentration, vacuum concentration, membrane concentration, etc., but are described in WO2010 / 067785, which uses less energy and can separate the fermentation inhibitor contained in the sugar solution. A concentrated sugar solution in which the sugar component is concentrated can be obtained by a method of filtering through a nanofiltration membrane and / or a reverse osmosis membrane.

  Various chemicals can be produced by growing microorganisms having the ability to produce chemicals using the sugar solution obtained by the present invention as a fermentation raw material. Here, growing a microorganism as a fermentation raw material means that a sugar component or an amino source contained in a sugar solution is used as a nutrient for the microorganism to propagate and maintain the growth of the microorganism. Specific examples of chemical products include substances that are mass-produced in the fermentation industry, such as alcohols, organic acids, amino acids, and nucleic acids. Such chemical products are accumulated and produced as chemical products inside and outside the body in the process of metabolism using the sugar component in the sugar solution as a carbon source. Specific examples of chemicals that can be produced by microorganisms include ethanol, 1,3-propanediol, 1,4-butanediol, glycerol and other alcohols, acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, itaconic acid, citric acid. Examples thereof include organic acids such as acids, nucleosides such as inosine and guanosine, nucleotides such as inosinic acid and guanylic acid, and amine compounds such as cadaverine. Furthermore, the sugar solution of the present invention can be applied to the production of enzymes, antibiotics, recombinant proteins and the like. The microorganism used for the production of such a chemical product may be any microorganism that can efficiently produce the target chemical product, and microorganisms such as Escherichia coli, yeast, filamentous fungi, and basidiomycetes can be used.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these.

(Reference Example 1) Preparation of cellulose-containing biomass Diluted sulfuric acid treatment of cellulose-containing biomass Cellulose-containing biomass (corn cob) was immersed in a 1% sulfuric acid aqueous solution and treated with an autoclave (manufactured by Nitto Koatsu) at 150 ° C for 30 minutes. After the treatment, solid-liquid separation was performed to separate into a solution component (hereinafter, dilute sulfuric acid treatment solution) and a treated biomass component (hereinafter, dilute sulfuric acid treatment product). The dilute sulfuric acid treated product was washed with pure water and washed away with sulfuric acid and used in the following examples.

2. Hydrothermal treatment of cellulose-containing biomass Cellulose-containing biomass (rice straw) was soaked in water and autoclaved at 180 ° C. for 20 minutes (manufactured by Nitto Koatsu) with stirring. The pressure at that time was 10 MPa. After the treatment, solid-liquid separation was performed to separate into a solution component (hereinafter, hydrothermally treated solution) and a treated biomass component (hereinafter, hydrothermally treated product).

Reference Example 2 Measurement of Sugar Concentration Glucose CII-Test Wako (manufactured by Wako Pure Chemical Industries) was used for measurement of glucose concentration, and D-XYLOSE ASSAY KIT (manufactured by Megazyme) was used for measurement of xylose concentration.

Reference Example 3 Preparation of Trichoderma genus cellulase Trichoderma genus cellulase was prepared by the following method.

[Pre-culture]
Corn steep liquor 5% (w / v), glucose 2% (w / v), ammonium tartrate 0.37% (w / v), ammonium sulfate 0.14% (w / v), potassium dihydrogen phosphate 14% (w / v), calcium chloride dihydrate 0.03% (w / v), magnesium sulfate heptahydrate 0.03% (w / v), zinc chloride 0.02% (w / v) ), Iron (III) chloride hexahydrate 0.01% (w / v), copper (II) sulfate pentahydrate 0.004% (w / v), manganese chloride tetrahydrate 0.0008% (W / v), boric acid 0.0006% (w / v), hexamolybdenum hexamolybdate tetrahydrate 0.026% (w / v) dissolved in distilled water, 100 mL of 500 mL baffle The mixture was placed in a conical flask and autoclaved at 121 ° C. for 15 minutes. After cooling, PE-M and Tween 80 that were autoclaved at 121 ° C. for 15 minutes were added to 0.01% (w / v). This preculture medium was inoculated with Trichoderma reesei PC3-7 at 1 × 10 ⁵ cells / mL and cultured with shaking at 28 ° C. and 180 rpm for 72 hours to obtain a preculture solution (shaking apparatus: TAITEC). BIO-SHAKER BR-40LF).

[Main culture]
Corn steep liquor 5% (w / v), glucose 2% (w / v), cellulose (Avicel) 10% (w / v), ammonium tartrate 0.37% (w / v), ammonium sulfate 0.14% ( w / v), potassium dihydrogen phosphate 0.2% (w / v), calcium chloride dihydrate 0.03% (w / v), magnesium sulfate heptahydrate 0.03% (w / v) ), Zinc chloride 0.02% (w / v), iron (III) chloride hexahydrate 0.01% (w / v), copper (II) sulfate pentahydrate 0.004% (w / v) ), Manganese chloride tetrahydrate 0.0008% (w / v), boric acid 0.006% (w / v), hexamolybdate hexaammonium tetrahydrate 0.0026% (w / v) Dissolved in distilled water, and put 2.5 L into a 5 L jar fermenter (DPC-2A manufactured by ABLE) Autoclaved at 121 ° C. for 15 minutes. After standing to cool, PE-M and Tween 80 autoclaved at 121 ° C. for 15 minutes each were added to 0.01% (w / v), respectively, and the preculture solution obtained by the above method was added. 250 mL was inoculated. Thereafter, the cells were cultured at 28 ° C., 300 rpm, and aeration volume 1 vvm for 87 hours. The obtained culture solution was used as a crude enzyme solution in the following examples.

(Reference example 4) Cellulase activity measuring method Cellulase activity was measured and evaluated by the following procedures for three types of 1) crystalline cellulose decomposition activity, 2) cellobiose decomposition activity, and 3) xylan decomposition activity.

1) Crystalline cellulose degradation activity A substrate solution was prepared by suspending crystal cellulose (Cellulose microcrystalline, manufactured by Merck) in 50 mM sodium acetate buffer (pH 5.2) to 1% by weight. To 500 μL of the substrate solution, 5 μL of the enzyme solution was added and reacted at 50 ° C. while rotating and mixing. The reaction time was basically 30 minutes, and was appropriately extended up to 60 minutes depending on the level of enzyme activity. After the reaction, the tube was centrifuged, and the glucose concentration of the supernatant component was measured. The glucose concentration was measured by the method described in Reference Example 2. The amount of enzyme that produces 1 μmol of glucose 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.
Crystalline cellulose degradation activity (U / mL) = glucose concentration (g / L) × 1000 × 505 (μL) / (180.16 × reaction time (minutes) × 5 (μL)).

2) Cellobiose degradation activity A solution of D (+)-cellobiose (manufactured by Wako Pure Chemical Industries, Ltd.) dissolved in 50 mM sodium acetate buffer (pH 5.2) to 15 mM was used as a substrate solution. To 500 μL of the substrate solution, 5 μL of the enzyme solution was added and reacted at 50 ° C. while rotating and mixing. The reaction time was basically 10 minutes and was appropriately extended up to 30 minutes depending on the level of enzyme activity. After the reaction, the tube was centrifuged, and the glucose concentration of the supernatant component was measured. The glucose concentration was measured by the method described in Reference Example 2. The amount of enzyme that produces 1 μmol of glucose 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.
Cellobiose degradation activity (U / mL) = glucose concentration (g / L) × 1000 × 505 (μL) / (180.16 × reaction time (min) × 5 (μL)).

3) Xylan Degradation Activity A substrate solution was prepared by suspending xylan (Xylan from Birchwood, manufactured by Fluka) in 50 mM sodium acetate buffer (pH 5.2) to 1% by weight. 5 μL of enzyme solution was added to 500 μL of the dispensed substrate solution, and reacted at 50 ° C. while rotating and mixing. The reaction time was basically 30 minutes, and was appropriately extended up to 60 minutes depending on the level of enzyme activity. After the reaction, the tube was centrifuged, and the xylose concentration of the supernatant component was measured. The xylose concentration was measured by the method described in Reference Example 2. The amount of enzyme that produces 1 μmol of xylose 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.
Xylan decomposition activity (U / mL) = xylose concentration (g / L) × 1000 × 505 (μL) / (150.13 × reaction time (min) × 5 (μL)).

(Comparative example 1) Recovery of cellulase from cellulose-containing biomass hydrolyzate As a comparative example, the activity of recovered cellulase when cellulase is recovered from a cellulose-containing biomass hydrolyzate not containing a fermentation residue of starch raw material is shown below.

(Step 1: hydrolysis of cellulose-containing biomass)
1 g each of the diluted sulfuric acid-treated product and hydrothermally treated product of cellulose-containing biomass were weighed into a 50 mL centrifuge tube, and ultrapure water was added so that the final concentration of the cellulose-containing biomass was 10% by weight. After 1 hour of rotation at 50 ° C. using a hybridization rotator (SN-06BN manufactured by Nisshin Rika), the pH was adjusted to a range of 5.0 to 6.0 using diluted sulfuric acid or diluted caustic soda. 5.6 mg of Trichoderma genus cellulase was added to this composition, and the mixture was subsequently rotated and mixed at 50 ° C. for 24 hours using a hybridization rotator.

(Step 2: Separation of cellulase component derived from filamentous fungus and sugar solution)
The hydrolyzate from step 1 was subjected to solid-liquid separation by centrifugation (8,000 G, 10 minutes) to obtain 7 g of supernatant and 3 g of hydrolysis residue. The hydrolysis residue was suspended again in about 7 mL of ultrapure water, and solid-liquid separated into 7 g of washing solution and 3 g of hydrolysis residue by centrifugation (8,000 G, 10 minutes). The supernatant of the hydrolyzate and the washing solution were combined and filtered using an ultrafiltration membrane with a molecular weight cut off of 10,000 (VIVASPIN 20 material: PES, manufactured by Sartorius Steadim Biotech). After centrifugal filtration at 8,000 G until the non-permeate side is 1 mL or less, ultrapure water is added to the non-permeate to dilute it 10 times or more for desalting, and again until the non-permeate side becomes 1 mL or less. Centrifugal filtration was performed at 000 G. The obtained non-permeate was used as a recovered enzyme solution, and activity was measured according to Reference Example 4.

(Comparative example 2) Effect of adding protein Zein (for biochemistry, manufactured by Wako Pure Chemical Industries, Ltd.) is the main protein of corn during hydrolysis of the dilute sulfuric acid-treated product and hydrothermally treated product of cellulose-containing biomass in Step 1 of Comparative Example 1. ) Was added in an amount of 10% based on the solid weight of the biomass. Other than that, a recovered enzyme solution was obtained in the same manner as in Comparative Example 1, and the activity was measured according to Reference Example 4.

(Comparative Example 3) Effect of adding bacterial cells In Step 1 of Comparative Example 1, during hydrolysis of the dilute sulfuric acid-treated product and hydrothermally treated product of cellulose-containing biomass, the yeast (Saccharomyces cerevisiae OC-2 strain) was transformed into biomass. An amount of 10% was added to the solid weight.

  Yeast was inoculated into YPD medium (yeast extract 1%, peptone 2%, glucose 2%) and cultured at 30 ° C. and 120 rpm for 24 hours. The pellet obtained by centrifuging the culture solution (8,000 G, 10 minutes) was used as yeast cells.

  A recovered enzyme solution was obtained in the same manner as in Comparative Example 1 except that yeast cells were added, and the activity was measured according to Reference Example 4.

(Example 1) Effect of adding fermentation residue of starch raw material In step 1 of Comparative Example 1, during the hydrolysis of the dilute sulfuric acid-treated product and hydrothermally treated product of cellulose-containing biomass, dry distilled cereal residue (DDG) of corn raw material (high Protein corn DDGS (manufactured by Wilbur Ellis) or sake lees (white crane special sake lees, made by Hakutsuru Sake Brewery) was added so that the solid weight was 10% of the solid weight of the biomass. Other than that, a recovered enzyme solution was obtained in the same manner as in Comparative Example 1, and the activity was measured according to Reference Example 4. About the result of Comparative Examples 1-3 and Example 1, the enzyme activity in the comparative example 1 was made into the reference | standard (1.0), and the relative activity was put together in Table 1. Addition of corn DDG increased the recovery of cellulase components involved in crystalline cellulose degrading activity and cellobiose degrading activity.

(Example 2) Influence of addition amount of fermentation residue of starch raw material In Example 1, corn DDG was added in amounts of 1, 5, 10, and 30% based on the solid weight of biomass. Other than that, a recovered enzyme solution was obtained in the same manner as in Comparative Example 1, and the activity was measured according to Reference Example 4. The enzyme activity in Comparative Example 1 was taken as the standard (1.0), and the relative activities are summarized in Table 2. The amount of cellulase components recovered increased at all levels in the test section, and the higher the amount of corn raw material distillation residue, the higher the effect.

(Comparative Example 4) Recovery of cellulase from cellulose-containing biomass hydrolyzate containing water-soluble inorganic salt In Step 1 of Comparative Example 1, sodium chloride was used as a solution for each of the 0. 1, 1, and 5% by weight were added. Other than that, a recovered enzyme solution was obtained in the same manner as in Comparative Example 1, and the activity was measured according to Reference Example 4.

(Example 3) Fermentation residue of starch raw material and addition effect of water-soluble inorganic salt In Example 1, 5% of corn DDG was added to the biomass solid weight during hydrolysis of the dilute sulfuric acid treated product of cellulose-containing biomass. Further, sodium chloride was added so as to be 0.1, 0.5, 1, 3.5, and 5% by weight, respectively, with respect to the weight of the reaction solution. Other than that, a recovered enzyme solution was obtained in the same manner as in Comparative Example 1, and activity was measured according to Reference Example 4. Regarding the results of Comparative Example 4 and Example 3, the enzyme activity in Comparative Example 1 was set as the standard (1.0), and the relative activities are summarized in Table 3. By further adding sodium chloride, which is a water-soluble inorganic salt, in addition to corn DDG, the recovered amount of cellulase components involved in cellobiose decomposition activity was dramatically increased.

(Example 4) Influence of kind of water-soluble inorganic salt in combination with fermentation residue of starch raw material In Example 3, potassium chloride, magnesium chloride, and ammonium sulfate were used instead of sodium chloride, and the addition concentration was 1 wt. %. Other than that, a recovered enzyme solution was obtained in the same manner as in Comparative Example 1, and the activity was measured according to Reference Example 4. The enzyme activity in Comparative Example 1 was taken as the standard (1.0), and the relative activities are summarized in Table 4. With any water-soluble inorganic salt, the recovered amount of cellulase component was hardly changed.

(Example 5) Fermentation residue of starch raw material and addition effect of polar organic solvent In Example 1, during hydrolysis reaction of a dilute sulfuric acid treatment product of cellulose-containing biomass, corn DDG is contained in an amount of 5% with respect to the solid weight of biomass. Further, ethylene glycol was added so as to be 0.1, 0.5, 1, 3.5, and 5% by weight, respectively, with respect to the weight of the reaction solution. Other than that, a recovered enzyme solution was obtained in the same manner as in Comparative Example 1, and the activity was measured according to Reference Example 4. The enzyme activity in Comparative Example 1 was taken as the standard (1.0), and the relative activities are summarized in Table 5. By further adding ethylene glycol, which is a polar organic solvent, in addition to corn DDG, the recovered amount of cellulase components involved in crystalline cellulose decomposition activity and cellobiose decomposition activity increased.

(Example 6) Influence of kind of polar organic solvent in combination with fermentation residue of starch raw material In Example 5, ethanol, isopropanol, and glycerol were used instead of ethylene glycol, and the addition concentration was 1 wt%. Other than that, a recovered enzyme solution was obtained in the same manner as in Comparative Example 1, and the activity was measured according to Reference Example 4. The enzyme activity in Comparative Example 1 was taken as the standard (1.0), and the relative activities are summarized in Table 6. The recovery of the cellulase component was almost the same regardless of which polar organic solvent was used.

(Example 7) Cleaning effect of hydrolysis residue by washing water containing water-soluble inorganic salt and alkali Hydrolysis reaction was carried out under conditions containing corn DDG in an amount of 5% of the solid weight of the cellulose-containing biomass diluted sulfuric acid. Then, the hydrolyzate was separated into 7 g of supernatant and 3 g of hydrolysis residue by centrifuging (8,000 G, 10 minutes). About 7 g of a 2% by weight aqueous sodium chloride solution was added to the hydrolysis residue as washing water and suspended. Further dilute caustic soda was added to the above suspension to adjust the pH to 7.5, and the mixture was rotated and mixed at 25 ° C. for 1 hour using a hybridization rotator, followed by centrifugation (8,000 G, 10 minutes). Separated to 3 g of hydrolysis residue. The supernatant and the washing solution were combined, and a recovered enzyme solution was obtained in the same manner as in Comparative Example 1, and the activity was measured according to Reference Example 4.

(Example 8) Washing effect of hydrolysis residue with washing water containing polar organic solvent In Example 7, 1 wt% aqueous glycerol solution was used instead of 2 wt% aqueous sodium chloride solution as washing water for the hydrolysis residue. Rotating and mixed for 1 hour at 50 ° C. Other than that, a recovered enzyme solution was obtained in the same manner as in Example 7, and the activity was measured according to Reference Example 4. The enzyme activity in Comparative Example 1 was taken as the standard (1.0), and the results of Example 7 and Example 8 are summarized in Table 7 as relative activities. By recovering the hydrolysis residue using washing water containing at least one of a water-soluble inorganic salt, an alkali, and a polar organic solvent, the recovered amount of cellulase components increased.

  The sugar liquid obtained in the present invention can be used as a sugar raw material for various fermentation products.

Claims (7)

The manufacturing method of the sugar liquid from cellulose containing biomass characterized by including the following processes (1) and (2).
Step (1): The cellulose-containing biomass is hydrolyzed by the filamentous fungus-derived cellulase under the condition containing the fermentation residue of the starch raw material in the range of 0.1 to 50% by weight with respect to the solid weight of the cellulose-containing biomass. Process,
Step (2): The solution component obtained by solid-liquid separation of the hydrolyzate in Step (1) is filtered through an ultrafiltration membrane, and the filamentous fungus-derived cellulase is recovered as a non-permeate and used as a permeate. A step of obtaining a sugar solution.   The filamentous fungus-derived cellulase in the step (1) contains one or more enzyme components selected from the group consisting of cellobiohydrase, endoglucanase, β-glucosidase, xylanase and β-xylosidase. The manufacturing method of the sugar liquid as described in any one of.   The method for producing a sugar liquid according to claim 1 or 2, wherein the fermentation residue of the starch raw material in step (1) is a distillation residue after ethanol fermentation.   The method for producing a sugar liquid according to any one of claims 1 to 3, wherein in the step (1), the dilute sulfuric acid-treated product or hydrothermally-treated product of cellulose-containing biomass is hydrolyzed.   The step (1) includes a water-soluble inorganic salt and / or a polar organic solvent in a concentration range of 0.1 to 5% by weight with respect to the total hydrolysis reaction amount. The manufacturing method of the sugar liquid as described in any one of. Furthermore, the manufacturing method of the sugar liquid in any one of Claim 1 to 5 characterized by including the following processes (3) and (4).
Step (3): A step of washing the hydrolysis residue obtained by solid-liquid separation in Step (2) with washing water containing one or more selected from the group consisting of a water-soluble inorganic salt, an alkali, and a polar organic solvent,
Step (4): A step of solid-liquid separation of the washed product of step (3) to separate a hydrolysis residue and a washing solution, and recovering a filamentous fungus-derived cellulase component contained in the washing solution. Production of a chemical product, characterized by fermenting and culturing a microorganism having an ability to produce a chemical product using the sugar solution obtained by the method for producing a sugar solution according to any one of claims 1 to 6 as a fermentation raw material. Method.

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