WO2014208493A1 - Method for producing sugar solution - Google Patents

Abstract

A method for producing a sugar solution according to the present invention is a method for producing the sugar solution form a cellulose-containing biomass, and comprises: (1) a step of hydrolyzing the cellulose-containing biomass with a filamentous fungus-originated cellulase to produce a hydrolysate; (2) a step of subjecting the hydrolysate to solid/liquid separation to produce a sugar solution and a glycosylation residue; (3) a step of washing the glycosylation residue with an aqueous solution containing a lignin blocking agent to produce a wash solution containing the filamentous fungus-originated cellulase contained in the glycosylation residue; and (4) a step of filtrating the wash solution to collect an unpenetrated solution containing the filamentous fungus-originated cellulase.

Description

Method for producing sugar solution

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

The fermentative production process of chemicals using sugar as a raw material is used for the production of various industrial raw materials. As the sugar used as the fermentation raw material, a process for efficiently producing a sugar solution from renewable non-edible resources, that is, cellulose-containing biomass, or a process for efficiently converting the obtained sugar solution as a fermentation raw material into an industrial raw material. Construction is under consideration.

As a method for producing a sugar solution from cellulose-containing biomass, a method for producing a sugar solution by hydrolyzing cellulose and hemicellulose with an acid using concentrated sulfuric acid (Patent Documents 1 and 2), and hydrolyzing a cellulose-containing biomass with dilute sulfuric acid. A method for producing a sugar solution by further performing saccharification treatment using a saccharifying enzyme such as cellulase after decomposition is disclosed (Non-patent Document 1). In addition, as a method not using an acid, a method of hydrolyzing cellulose-containing biomass using subcritical water at about 250 to 500 ° C. to produce a sugar solution (Patent Document 3), a method of using cellulose-containing biomass with subcritical water After the treatment, a method for producing a sugar solution by further saccharifying with a saccharifying enzyme (Patent Document 4), hydrolyzing cellulose-containing biomass with hot hot water at 240 to 280 ° C., and further using a saccharifying enzyme A method for producing a sugar solution by saccharification treatment (Patent Document 5) is disclosed. Among these, in recent years, a method for hydrolyzing biomass using a saccharifying enzyme that has a particularly small amount of energy consumption and an environmental load and that has a high sugar yield has been widely studied. However, the method for producing a sugar solution using such a saccharifying enzyme has a high cost for the enzyme, and therefore the cost for producing the sugar solution is high.

Therefore, as a method for solving the above technical problem, a method for recovering and reusing the saccharifying enzyme used for hydrolysis has been proposed. For example, a solid-liquid separation is carried out continuously with a spin filter, the obtained sugar solution is filtered through an ultrafiltration membrane, and the enzyme is recovered (Patent Document 6). In the stage of enzymatic saccharification, cellulose is converted into an enzyme. A method for improving the recovery efficiency by introducing a surfactant into the solid content remaining in the saccharified solution saccharified in (Patent Document 7), and conducting the saccharification residue after enzymatic saccharification A method for recovering an enzyme component (Patent Document 8), a method for recovering and reusing an enzyme by putting the saccharification residue after enzymatic saccharification into new biomass again (Patent Document 9), and the like are disclosed. .

Japanese National Patent Publication No. 11-506934 JP 2005-229821 A Japanese Patent Laid-Open No. 2003-212888 JP 2001-95597 A Japanese Patent No. 3041380 JP 2006-87319 A JP-A-63-87994 JP 2008-206484 A Japanese Patent Application Laid-Open No. 55-144885

As described above, a method for hydrolyzing cellulose-containing biomass using a saccharifying enzyme has been developed. From the viewpoint of reducing the amount of saccharifying enzyme used, a saccharifying enzyme is used for producing a sugar solution from cellulose-containing biomass. There is a need for a method for producing a sugar solution while using it more effectively.

Therefore, in view of such circumstances, an object of the present invention is to provide a method for producing a sugar solution that can produce a sugar solution while further reducing the amount of saccharifying enzyme used.

In order to solve the above-described problems, the present inventors have intensively studied a method for producing a sugar solution. As a result, the filamentous fungus-derived cellulase adsorbed on the saccharification residue is eluted into an aqueous solution containing a lignin blocking agent, and the aqueous solution from which the filamentous fungus-derived cellulase is eluted is filtered to obtain a non-permeate containing the filamentous fungus-derived cellulase. It was found that a sugar solution can be produced by further reducing the amount of filamentous fungus-derived cellulase used by collecting and reusing. The present invention has been completed based on such findings.

The present invention has the following configurations [1] to [10].
[1] A method for producing a sugar solution from cellulose-containing biomass,
Step (1): A step of hydrolyzing the cellulose-containing biomass with a filamentous fungus-derived cellulase to obtain a hydrolyzate,
Step (2): Solid-liquid separation of the hydrolyzate into a sugar solution and a saccharification residue,
Step (3): washing the saccharification residue with an aqueous solution containing a lignin blocking agent to obtain a washed solution containing the filamentous fungus-derived cellulase contained in the saccharification residue, and step (4): the washing Filtering the solution containing the solution, and recovering the non-permeate containing the filamentous fungus-derived cellulase,
A method for producing a sugar solution, comprising:
[2] The lignin blocking agent is corn steep liquor, peptone, yeast extract, meat extract, casein, bovine serum-derived albumin, skim milk, ethanol fermentation distillation residue, zein, fish processing waste, meat processing waste, whey protein, The method for producing a sugar liquid according to [1], wherein the sugar solution is one or more selected from the group consisting of grain processing waste, sugar processing waste, food, algal protein, soy protein, bacterial protein, and fungal protein.
[3] The method for producing a sugar solution according to [1] or [2], wherein in the step (4), filtration of the washed solution is an ultrafiltration membrane.
[4] In the step (4), the method includes a step of mixing the sugar solution obtained in the step (2) with the washed solution obtained in the step (3), and the sugar solution and the washed solution The method for producing a sugar solution according to any one of [1] to [3], wherein a mixed solution containing the solution is filtered to collect a permeate containing the sugar solution.
[5] The method for producing a sugar liquid according to any one of [1] to [4], wherein in step (3), the temperature of the aqueous solution containing the lignin blocking agent is 40 to 60 ° C.
[6] The method for producing a sugar liquid according to any one of [1] to [5], wherein the aqueous solution containing the lignin blocking agent contains an inorganic salt.
[7] The method for producing a sugar liquid according to any one of [1] to [6], wherein the filamentous fungus-derived cellulase is derived from a Trichoderma microorganism.
[8] The method for producing a sugar liquid according to any one of [1] to [7], wherein the non-permeating liquid is mixed with the filamentous fungus-derived cellulase in step (1).
[9] The method for producing a sugar liquid according to any one of [1] to [8], wherein in the step (2), the solid-liquid separation of the hydrolyzate is performed by press filtration.
[10] A step of producing a sugar solution by the method for producing a sugar solution according to any one of [1] to [9];
Culturing a microorganism having the ability to produce a chemical using the sugar solution as a fermentation raw material;
A method for producing a chemical product.

According to the present invention, the filamentous fungus-derived cellulase adsorbed on the saccharification residue is eluted in an aqueous solution containing a lignin blocking agent, and the aqueous solution from which the filamentous fungus-derived cellulase is eluted is filtered, thereby impermeable liquid containing the filamentous fungus-derived cellulase. Can be obtained. By collecting and reusing the obtained non-permeate containing the filamentous fungus-derived cellulase, a sugar solution can be produced while further reducing the amount of the filamentous fungus-derived cellulase used. Thereby, the manufacturing cost of a sugar liquid can be reduced significantly.

It is a figure which shows the manufacturing method of the sugar liquid by this invention. It is a figure which shows another example of the manufacturing method of the sugar liquid by this invention. It is a figure which shows an example of the sugar liquid manufacturing apparatus using the manufacturing method of the sugar liquid by this invention.

Hereinafter, embodiments for carrying out the present invention will be described in detail. In addition, the form for implementing this invention is not limited to the following.

<Method for producing sugar solution>
A method for producing a sugar solution according to the present invention is shown in FIG. Hereinafter, the method for producing a sugar solution according to the present invention will be described for each step.

[Step (1)]
In the step (1), a filamentous fungus-derived cellulase that is a saccharifying enzyme is added to the cellulose-containing biomass, and the cellulose-containing biomass is hydrolyzed by the filamentous fungus-derived cellulase. Thereby, a hydrolyzate is obtained. The purpose of hydrolysis is to reduce the molecular weight of cellulose to produce monosaccharides or oligosaccharides, but in the hydrolysis of cellulose-containing biomass, hemicellulose components such as xylan, mannan and arabinan are also hydrolyzed at the same time.

Cellulose-containing biomass refers to biological resources that contain cellulose components. Specifically, herbaceous biomass such as bagasse, switchgrass, napiergrass, Eliansus, corn stover, beet pulp, cottonseed husk, palm husk, rice straw, straw, bamboo, straw, birch, beech, etc. Woody biomass such as wood and waste building materials, as well as biomass derived from the aquatic environment such as algae and seaweed. The cellulose-containing biomass contains lignin, which is an aromatic polymer, in addition to cellulose composed of sugar and hemicellulose (hereinafter referred to as “cellulose” as a generic term for cellulose and hemicellulose).

The reaction conditions for the hydrolysis with a saccharifying enzyme may be carried out in accordance with the preferable reaction conditions for a saccharifying enzyme. In the present invention, since a filamentous fungus-derived cellulase is used, the reaction temperature is preferably in the range of 15 to 100 ° C. 40 to 60 ° C is more preferable, and around 50 ° C is more preferable.

The reaction time for hydrolysis is preferably in the range of 2 hours to 200 hours. If it is 2 hours or more, sufficient sugar is produced. Moreover, if it is 200 hours or less, the fall of enzyme activity can be suppressed and the collect | recovered saccharifying enzyme can be reused.

The pH of the hydrolysis reaction has the highest effect of hydrolysis by the filamentous fungus-derived cellulase at the optimum pH of the filamentous fungus-derived cellulase, so the pH during the cellulase treatment should be the optimum pH of the filamentous fungus-derived cellulase. preferable. In the present invention, since filamentous fungus-derived cellulase is used, the pH is preferably in the range of 3 to 9, more preferably 4 to 5.5, and even more preferably around 5. When Trichoderma genus cellulase is used as the filamentous fungus-derived cellulase, the optimum pH for the reaction is 5.0.

Adjustment of the pH of the reaction solution containing cellulose-containing biomass and filamentous fungus-derived cellulase is performed immediately before solid-liquid separation of the hydrolyzate or simultaneously with solid-liquid separation of the hydrolyzate in step (2) described later. May be. The pH can be adjusted before solid-liquid separation of the hydrolyzate, and the effect can be further enhanced by allowing it to stand for a certain period of time until solid-liquid separation is started. For example, after adjusting the pH of the hydrolyzate, there is a method of solid-liquid separation after standing for 1 hour.

Furthermore, since a change in pH occurs during the hydrolysis process, it is preferable to adjust the pH so that the pH becomes constant using acid or alkali. Moreover, pH adjustment may use a buffer solution for a hydrolyzate suitably. The acid or alkali used for pH adjustment is not particularly limited. Examples of the acid include hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid and the like. Preferably, sulfuric acid, nitric acid, and phosphoric acid are used from the viewpoint that inhibition during fermentation of the sugar liquid obtained in the present invention hardly occurs. More preferably, sulfuric acid is used from the viewpoint of economy. As the alkali, preferably, ammonia, sodium hydroxide, calcium hydroxide and an aqueous solution containing them are used from the viewpoint of economy, and more preferably, membrane fouling is performed during membrane separation in step (4) described later. From the viewpoint of suppressing the occurrence of water, monovalent ions such as ammonia and sodium hydroxide are used, and more preferably, ammonia is used from the viewpoint that inhibition during fermentation hardly occurs.

In order to promote the contact between the cellulose-containing biomass and the saccharifying enzyme, and to make the sugar concentration of the hydrolyzate uniform, it is preferable to mix the cellulose-containing biomass and the saccharifying enzyme while stirring.

The solid content concentration of cellulose is preferably 1 to 25% by weight, more preferably 5 to 20% by weight.

As the cellulase derived from filamentous fungi used as a saccharifying enzyme, the genus Trichoderma, Aspergillus, Cellulomonas, Clostridium, Streptomyces, Humi, Examples include cellulases derived from microorganisms such as Acremonium, Irpex, Mucor, Talaromyces, Phanerochaete, white rotten gold, and brown rotting fungi. . In addition, a cellulase derived from a mutant strain in which cellulase productivity has been improved by subjecting these microorganisms to a mutation treatment or irradiation with ultraviolet rays or the like may be used. Among these filamentous fungus-derived cellulases, it is preferable to use Trichoderma genus-derived cellulase, which produces a large amount of enzyme components with high specific activity in the hydrolysis of cellulose.

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 exemplified Trichoderma viride QM9123 a (Trichoderma viride9123).

Filamentous fungus-derived cellulase is an enzyme composition that contains a plurality of enzymes that hydrolyze cellulose and has an activity of hydrolyzing and saccharifying cellulose and / or hemicellulose. Examples of enzymes that hydrolyze cellulose include cellobiohydrase, endoglucanase, exoglucanase, β-glucosidase, xylanase, and xylosidase. Since the filamentous fungus-derived cellulase contains a plurality of such enzymes, cellulose and / or hemicellulose can be efficiently hydrolyzed by the concerted effect or the complementary effect of the plurality of enzymes in cellulose degradation.

In particular, the filamentous fungus-derived cellulase used in the present invention preferably contains cellobiohydrase and xylanase. In the present invention, by adding a high-concentration inorganic salt to cellulose-containing biomass and filamentous fungus-derived cellulase at the time of hydrolysis of cellulose-containing biomass, cellobio for the pretreatment product of biomass before hydrolysis of cellulose-containing biomass is performed. This is because adsorption of hydrase and xylanase is reduced and a high enzyme recovery rate is obtained.

Cellobiohydrase is a general term for cellulases that start hydrolysis of cellulose from the terminal portion and release cellobiose, and an enzyme group belonging to cellobiohydrase as EC number: EC 3.2.1.91. Is described. Cellulolytic activity can be measured from the amount of glucose liberated when an enzyme is allowed to act on cellulose as a substrate, and specific methods are described in “Pure & Appl. Chem., Vol. 59, No. 2”. 257-268 page, "FILTER PAPER ASSAY FOR SACCHARIFYING CELLULASE" can be used.

Endoglucanase is a general term for cellulases having an activity of hydrolyzing from the central part of a cellulose molecular chain, and EC numbers: EC 3.2.1.4, EC 3.2.1.6, EC 3.2.1.39. EC 3.2.1.73 describes an enzyme group belonging to endoglucanase. Cellulolytic activity can be measured from the amount of reducing sugar released when an enzyme is allowed to act using carboxymethylcellulose (CMC) as a substrate. Specific methods are described in, for example, “Pure & Appl. Chem. , Vol.59, No.2, pp. 257-268 ”, the method described in“ CARBOXYL CELLULASE ASSAY FOR ENDO-β-1,4-GLUCANCE ”can be used.

Exoglucanase is a general term for cellulases that hydrolyze from the ends of cellulose molecular chains, and the enzyme groups belonging to exoglucanase are described as EC numbers: EC3.2.1.74 and EC3.2.1.58. ing.

Β-glucosidase is a general term for cellulases that hydrolyze cellooligosaccharides or cellobiose, and describes an enzyme group belonging to β-glucosidase as EC number: EC 3.2.1.21. Cellobiose degrading activity (hereinafter also referred to as “BGL activity”) can be measured from the amount of glucose released when an enzyme is allowed to act on cellobiose as a substrate. For example, “Pure & Appl. Chem. Vol.59, No.2, pages 257-268 "can be measured according to the method of" Cellobiase assay ".

Xylanase is a general term for cellulases 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 cellulases characterized by acting on xylo-oligosaccharides, and an enzyme group belonging to xylosidase is described as EC number: EC 3.2.1.37.

Enzymes contained in such filamentous fungus-derived cellulases are separated by known techniques such as gel filtration, ion exchange, two-dimensional electrophoresis, and the amino acid sequences of the separated components (N-terminal analysis, C-terminal analysis, mass spectrometry) are performed. It can be identified by comparison with a database.

In addition, the enzyme activity of filamentous fungus-derived cellulase can be evaluated by polysaccharide hydrolysis activity such as Avicel degradation activity, xylan degradation activity, carboxymethylcellulose (CMC) degradation activity, cellobiose degradation activity, mannan degradation activity. The main enzyme exhibiting Avicel-degrading activity is cellobiohydrase or exoglucanase, which has the characteristic of hydrolyzing from the terminal portion of cellulose. The main enzymes showing xylan degradation activity are xylanase and β-xylosidase. The main enzymes involved in CMC degradation activity are cellobiohydrase, exoglucanase and endoglucanase. The main enzyme exhibiting cellobiose degrading activity is β-glucosidase. 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, a crude enzyme product is preferably used as the cellulase derived from filamentous fungi. The crude enzyme product is derived from the culture supernatant obtained by culturing the microorganism for an arbitrary period in a medium adjusted so that the microorganism of the genus Trichoderma 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. As the crude enzyme product, the culture supernatant is preferably used as it is or from the culture supernatant obtained by removing Trichoderma cells.

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, etc. are contained in the components. Trichoderma microorganisms produce a strong cellulase component in the culture solution, while β-glucosidase has low β-glucosidase activity in the culture solution because it is retained in the cell or on the cell surface. Therefore, you may add a different type or the same type of beta-glucosidase to the crude enzyme product. As the heterogeneous β-glucosidase, β-glucosidase derived from Aspergillus can be preferably used. Examples of β-glucosidase derived from the genus Aspergillus include Novozyme 188 commercially available from Novozyme. As a method of adding a heterologous or homologous β-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.

(Preprocessing)
Cellulose-containing biomass contains lignin and the like in addition to cellulose as described above. Therefore, it is preferable to pretreat the cellulose-containing biomass. Thereby, the hydrolysis efficiency of the cellulose containing biomass by a filamentous fungus origin cellulase can be improved. Examples of the pretreatment method for cellulose-containing biomass include acid treatment, sulfuric acid treatment, dilute sulfuric acid treatment, acetic acid treatment, alkali treatment, caustic soda treatment, ammonia treatment, hydrothermal treatment, subcritical water treatment, pulverization treatment, and steaming treatment. . In the present invention, it is preferable to perform ammonia treatment, hydrothermal treatment or dilute sulfuric acid treatment from the viewpoint of efficiently recovering various enzymes in the step (3) described later.

The ammonia treatment can be performed according to the methods described in JP 2008-161125 A, JP 2008-535664 A, and the like. For example, the ammonia concentration to be used is added in the range of 0.1 to 15% by weight with respect to the biomass, and the treatment is performed at 4 to 200 ° C., preferably 90 to 150 ° C. Ammonia to be added may be either liquid or gas. The form to be added may be pure ammonia or an aqueous ammonia solution. 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. Since the treated product obtained by the ammonia treatment is further subjected to an enzymatic hydrolysis reaction, it is necessary to neutralize ammonia or remove ammonia. Neutralization may be performed on ammonia from which the solid content has been removed from the hydrolyzate by solid-liquid separation, or may be performed while the solid content is still contained. The acid reagent used for neutralization is not particularly limited. Ammonia can be removed by volatilizing ammonia into a gaseous state by keeping the ammonia-treated product in a reduced pressure state. The removed ammonia may be recovered and reused.

In the case of hydrothermal treatment, after adding water so that the cellulose-containing biomass becomes 0.1 to 50% by weight, it is treated at a temperature of 100 to 400 ° C. for 1 second to 60 minutes. By treating under such temperature conditions, hydrolysis of cellulose occurs. 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.

In the case of dilute sulfuric acid treatment, the concentration of sulfuric acid is preferably 0.1 to 15% by weight, and more preferably 0.5 to 5% by weight. The reaction temperature can be set in the range of 100 to 300 ° C., preferably 120 to 250 ° C. The reaction time can be set in the range of 1 second to 60 minutes. The number of processes is not particularly limited, and the process may be performed once or more. In particular, when the above process is performed twice or more, the first process and the second and subsequent processes may be performed under different conditions. The hydrolyzate obtained by the dilute sulfuric acid treatment contains an acid, and further needs to be neutralized in order to perform a hydrolysis reaction with cellulase or to use as a fermentation raw material.

[Step (2)]
In step (2), the hydrolyzate obtained in step (1) is subjected to solid-liquid separation into a sugar solution and a saccharification residue.

The method for solid-liquid separation of the hydrolyzate in the step (2) is not particularly limited, and a conventionally known general solid-liquid separation method can be used. Examples of the solid-liquid separation method include centrifugal separation using a screw decanter, membrane separation such as a filter press, separation using a belt filter, natural sedimentation, or filtration using a mesh screen, nonwoven fabric, filter paper, and the like. As the solid-liquid separation method of the hydrolyzate, it is preferable to use membrane separation. Among membrane separations, the hydrolyzate solid-liquid separation method can efficiently remove the saccharification residue, which is a particulate solid, and can recover more saccharose by pressing the removed saccharification residue. From the viewpoint that the hydrolyzate can be solid-liquid separated by press filtration, it is most preferable to use a filter press. The method for solid-liquid separation of the hydrolyzate may be used singly or in combination.

[Step (3)]
In the step (3), the saccharification residue is washed with an aqueous solution containing a lignin blocking agent, and the filamentous fungus-derived cellulase contained by adsorption to the saccharification residue is eluted into the aqueous solution containing the lignin blocking agent. As a result, a washed solution containing the filamentous fungus-derived cellulase contained in the saccharification residue is obtained.

The lignin blocking agent is a component that has an affinity for the lignin component contained in the cellulose-containing biomass and can be adsorbed by hydrophobic interaction, hydrogen bond, ionic bond, and the like. Specific examples of the component include hydrophilic polymer compounds such as peptides, proteins, and polysaccharides. In addition, the lignin blocking agent is derived from filamentous fungus adsorbed on the saccharification residue because it contains other components such as sugars, amino acids, salts, fats, etc. than those containing these polymer compounds alone. This is preferable from the viewpoint of enhancing the elution effect of cellulase.

Specific examples of preferable lignin blocking agents containing the polymer compound and other components include corn steep liquor (CSL), peptone, yeast extract, meat extract, casein, skim milk, bovine serum-derived albumin (BSA), ethanol fermentation Distilled residue (DDGS), zein, fish processing waste, meat processing waste, whey protein (whey protein), sugar processing waste, grain processing waste, food, algal protein, soy protein, bacterial protein, fungal protein, etc. Is mentioned. These may be used alone or as a mixture of two or more. Among these, a coastal liquor and / or ethanol fermentation distillation residue is more preferable. Moreover, the lignin blocking agent exemplified above has an advantage that the price per unit is low.

The lignin blocking agent is preferably contained in the aqueous solution at a concentration of 1 g / L or more from the viewpoint of enhancing the elution effect of the filamentous fungus-derived cellulase adsorbed on the saccharification residue, and more preferably 1 to 10 g / L. preferable. Note that the lignin blocking agent contained in the aqueous solution need not be in a state of being completely dissolved in water, but may be partially insolubilized.

It is preferable that the aqueous solution containing the lignin blocking agent further contains an inorganic salt. Thereby, an enzyme component that cannot be eluted only with a lignin blocking agent, specifically, a hemicellulase component such as xylosidase can be eluted.

Examples of inorganic salts include sodium chloride, sodium sulfate, magnesium chloride, magnesium sulfate, calcium chloride, and ammonium sulfate. Among these, ammonium sulfate is preferable. The concentration of the inorganic salt contained in the aqueous solution containing the lignin blocking agent is preferably 1 to 25 g / L.

The washing of the saccharification residue may be a method of dispersing the saccharification residue in an aqueous solution containing a lignin blocking agent and stirring and mixing, or a method of passing an aqueous solution containing a lignin blocking agent through the saccharification residue. From the viewpoint of enhancing the elution effect of the filamentous fungus-derived cellulase adsorbed on the saccharification residue, a method of passing an aqueous solution containing a lignin blocking agent to the saccharification residue is preferable.

When the saccharification residue is washed with an aqueous solution containing a lignin blocking agent, the temperature of the aqueous solution containing the lignin blocking agent is preferably 40 to 60 ° C. When the temperature at the time of washing is 40 ° C. or higher, the washing effect of the saccharification residue is increased, and the amount of filamentous fungus-derived cellulase eluted is increased. When the temperature at the time of washing is 60 ° C. or lower, it is possible to suppress the inactivation of the filamentous fungus-derived cellulase, so that it is possible to suppress the reduction of each activity of the filamentous fungus-derived cellulase in the washed solution. it can.

[Step (4)]
In step (4), the washed solution is filtered and separated into a permeate containing a sugar solution and a non-permeate containing a filamentous fungus-derived cellulase. As a result, the permeate is collected and the non-permeate containing the filamentous fungus-derived cellulase is collected. Since the permeate contains a sugar liquid, it can be used as a sugar liquid by being mixed with the sugar liquid obtained in the step (2).

For filtration of the washed solution, a method is used that allows permeation of monosaccharides such as glucose (molecular weight 180) and xylose (molecular weight 150) and prevents permeation of filamentous fungus-derived cellulase.

In the present invention, an ultrafiltration membrane (UF membrane) is preferably used for filtering the washed solution. The ultrafiltration membrane can permeate sugar while preventing permeation of macromolecules such as filamentous fungus-derived cellulase contained in the washed solution. Filamentous fungal cellulase adsorbed on the saccharification residue is eluted in the washed solution obtained by washing the saccharification residue. Filtration of the washed solution through an ultrafiltration membrane separates and recovers the filamentous fungus-derived cellulase contained in the washed solution from the washed solution as an ultrafiltration membrane non-permeate. Can do. Further, the washed solution may be filtered through an ultrafiltration membrane sequentially or continuously.

As the ultrafiltration membrane, it is preferable to use a membrane having a fractional molecular weight capable of permeating monosaccharides such as glucose and xylose and preventing permeation of filamentous fungus-derived cellulase. In the present invention, the molecular weight cutoff of the ultrafiltration membrane is preferably 500 to 100,000. If an ultrafiltration membrane having a molecular weight cut-off within this range is used, it is possible to permeate monosaccharides as a sugar solution while preventing permeation of filamentous fungus-derived cellulase. Further, the molecular weight cut off of the ultrafiltration membrane is more preferably 10,000 to 50,000 from the viewpoint of separating contaminants having an inhibitory action on the enzyme reaction from the enzyme.

Here, the ultrafiltration membrane is too small to measure the pore diameter on the membrane surface with an electron microscope or the like, and instead of the average pore diameter, the value of the fractional molecular weight is an index of the pore size. It is supposed to be. The molecular weight cut-off refers to the Membrane Science Experiment Series Vol. III, Artificial Membrane Editor, Editorial Committee / Naofumi Kimura, Shinichi Nakao, Haruhiko Ohya, Tsutomu Nakagawa (1993, Kyoritsu Shuppan), P92. A plot of data with the rejection rate on the vertical axis is called a fractional molecular weight curve. The molecular weight at which the blocking rate is 90% is called the fractional molecular weight of the membrane. ”Is well known to those skilled in the art as an index representing the membrane performance of the ultrafiltration membrane.

Examples of the ultrafiltration membrane used include polyethersulfone (PES), polysulfone (PS), polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), regenerated cellulose, cellulose, cellulose ester, polysulfone, polyethersulfone, Organic materials such as chlorinated polyethylene, polypropylene, sulfonated polysulfone, sulfonated polyethersulfone, polyolefin, polyvinyl alcohol, polymethyl methacrylate, polytetrafluoroethylene, metals such as stainless steel, or inorganic materials such as ceramic . As the material of the ultrafiltration membrane to be used, it is preferable to use PES, PVDF or the like because regenerated cellulose, cellulose, and cellulose ester are decomposed by cellulase.

As the form of the ultrafiltration membrane, a tubular type, a spiral type, a flat membrane type, a hollow fiber type and the like can be preferably used. Specifically, DESAL's 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, SPE1 from Sinder, SPE3, SPE5, SPE10, SPE30, SPV5, SPV50, SOW30, Microza from 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.

Examples of the ultrafiltration membrane filtration method include a crossflow filtration method and a dead-end filtration method, but the crossflow filtration method is preferable from the viewpoint of suppressing membrane fouling and flux.

As the filtration method, pressure filtration, vacuum filtration, centrifugal filtration and the like can be preferably used. Examples of the filtration operation include constant pressure filtration, constant flow filtration, non-constant pressure non-constant flow filtration, and the like. The filtration operation may be multistage filtration using the ultrafiltration membrane twice or more.

Since the non-permeated liquid recovered in the step (4) contains a filamentous fungus-derived cellulase, the recovered non-permeated liquid is mixed with the filamentous fungus-derived cellulase used in the step (1). It can be used as the filamentous fungus-derived cellulase of (1). Thereby, since the amount of the filamentous fungus-derived cellulase newly used in the step (1) can be reduced, the cost of the filamentous fungus-derived cellulase can be reduced.

Thus, according to the method for producing a sugar solution of the present invention, by eluting the filamentous fungus-derived cellulase adsorbed on the saccharification residue into an aqueous solution containing a lignin blocking agent, and filtering the aqueous solution from which the filamentous fungus-derived cellulase is eluted, A non-permeate liquid containing a filamentous fungus-derived cellulase can be obtained. By collecting and reusing the filamentous fungus-derived cellulase in the obtained non-permeate, a sugar solution can be produced while further reducing the amount of saccharifying enzyme used. Thereby, the manufacturing cost of a sugar liquid can be reduced significantly.

(Other forms)
In the present invention, the case where the liquid washed in the step (4) is filtered and the obtained non-permeated liquid is recovered has been described, but the present invention is not limited to this. For example, the sugar solution obtained in the step (2) is filtered separately from the washed solution to separate the filamentous fungus-derived cellulase contained in the sugar solution from the sugar solution, and the permeate is recovered as the sugar solution, You may make it collect | recover the non-permeated liquid containing a fungal origin cellulase. Thereby, the filamentous fungus-derived cellulase contained in the sugar solution can also be effectively reused.

In addition, after the sugar solution obtained in step (2) and the washed solution obtained in step (3) are mixed, the mixed solution is filtered to separate the cellulase derived from filamentous fungi contained in the mixed solution. You may make it do. Thereby, while being able to collect | recover the permeate containing a sugar liquid, the non-permeate containing a filamentous fungus origin cellulase can be collect | recovered. An example of the form which filters a mixed solution is shown in FIG. As shown in FIG. 2, the sugar solution and the washed solution are mixed to obtain a mixed solution. Thereafter, in step (4), the mixed solution is filtered through an ultrafiltration membrane, and separated into a non-permeate containing cellulase derived from filamentous fungi and a permeate containing a sugar solution. Thereby, the permeate containing the sugar solution is recovered and the non-permeate containing the cellulase derived from the filamentous fungus is recovered. The permeate may be used as a sugar solution as it is, or only a sugar solution obtained by separating components other than the sugar solution may be used. The recovered non-permeate can be mixed with the filamentous fungus-derived cellulase added in step (1) and reused in step (1). Although most of the filamentous fungus-derived cellulase is adsorbed to the saccharification residue obtained in the step (2), the sugar-containing biomass obtained in the step (2) also hydrolyzes the cellulose-containing biomass in the step (1). Contains the cellulase component derived from the filamentous fungus used. Therefore, in the step (4), the filamentous fungus-derived cellulase contained in the sugar solution obtained in the step (2) can also be collected together with the filamentous fungus-derived cellulase contained in the washed solution. The amount of cellulase recovered can be further increased. This makes it possible to produce a sugar solution while further reducing the amount of saccharifying enzyme used. In addition, compared with the case where the sugar solution obtained in step (2) is filtered separately from the washed solution, the case where the mixed solution is filtered is more efficient for the production of cellulase derived from filamentous fungi while simplifying the production apparatus. It can be recovered well.

In addition, the mixed solution may be prepared by temporarily holding the sugar solution obtained in step (2) before filtering and mixing it with the washed solution obtained in step (3). preferable. In general, proteins tend to be adsorbed on the surface of an ultrafiltration membrane, and can also be adsorbed to pipes, tanks, and the like. Since the main protein component contained in the sugar solution obtained in the step (2) is a filamentous fungus-derived cellulase component, if the protein component is adsorbed in the ultrafiltration membrane, piping, or tank, it is recovered. The amount of cellulase derived from bacteria will decrease. Therefore, the sugar solution in the step (2) and the washed solution obtained in the step (3) are mixed in advance, and the mixed solution is filtered through a filtration membrane such as an ultrafiltration membrane, whereby the sugar is obtained. It can suppress that the filamentous fungus origin cellulase component in a liquid adsorb | sucks especially in an ultrafiltration membrane, piping, a tank inside. Thereby, the collection amount of the filamentous fungus-derived cellulase can be further increased.

<Sugar solution production equipment>
A sugar solution production apparatus using the method for producing a sugar solution according to the present invention will be described. In addition, the form of the sugar liquid manufacturing apparatus is not limited to the following. FIG. 3 is a diagram showing an example of a sugar liquid production apparatus using the sugar liquid production method according to the present invention. FIG. 3 shows an apparatus using the method for producing a sugar solution according to the present invention described in FIG. As shown in FIG. 3, a sugar solution production apparatus 10 using the method for producing a sugar solution according to the present invention includes a hydrolysis reaction tank 11, a solid-liquid separator 12, a washing water tank 13, a filtrate collection tank 14, and ultrafiltration. It has a membrane device 15 and a saccharification enzyme recovery line L11.

The hydrolysis reaction tank 11 includes a stirring tank 21 that performs hydrolysis, a stirring device 23 that stirs and mixes the cellulose-containing biomass 22, and a heat retaining device 24 that keeps the stirring tank 21 warm. The stirring tank 21 includes a supply port 25 to which the cellulose-containing biomass 22 is supplied and a supply port 27 to which a filamentous fungus-derived cellulase 26 is supplied. When the cellulose-containing biomass 22 and the filamentous fungus-derived cellulase 26 are supplied into the stirring tank 21, the cellulose-containing biomass 22 is hydrolyzed by the filamentous fungus-derived cellulase 26 in the stirring tank 21 to obtain a hydrolyzate 28 ( Step (1)).

The hydrolyzate 28 obtained in the stirring tank 21 is extracted from the stirring tank 21 by opening the control valve V11, is pumped by the pump P1, and is supplied to the solid-liquid separator 12 from the supply port 31.

The solid-liquid separator 12 includes a press filtration apparatus 32 and a compressor 33. The hydrolyzate 28 is squeezed in the press filtration apparatus 32 by the compressor 33, so that it is separated into a liquid and a saccharification residue (solid) (step (2)). The sugar liquid is discharged from the press filtration apparatus 32 to the sugar liquid supply line L21, and the saccharification residue is retained in the filtration chamber.

In the middle of the sugar solution supply line L21, a branch line L22 connected to the washing water tank 13 is connected. The sugar solution supply line L21 is provided with a control valve V21, and the branch line L22 is provided with a control valve V22. By opening the control valve V21 and closing the control valve V22, the sugar solution discharged from the press filtration apparatus 32 is supplied to the filtrate collection tank 14 through the sugar solution supply line L21, and the filtrate collection tank. 14. Also, by closing the control valve V21 and opening the control valve V22, the sugar liquid discharged from the press filtration apparatus 32 is supplied to the washing water tank 13 through the branch line L22 and held in the washing water tank 13. Is done.

The aqueous solution 34 containing the lignin blocking agent is supplied from the cleaning water tank 13 through the cleaning liquid supply line L23 to the press filtration apparatus 32 through the water inlet 35. The saccharification residue generated in the press filtration apparatus 32 is washed with the aqueous solution 34 containing the lignin blocking agent, and the filamentous fungus-derived cellulase adhering to the saccharification residue is eluted into the aqueous solution 34 containing the lignin blocking agent, and is derived from the filamentous fungus. A washed solution containing cellulase is obtained (step (3)). The aqueous solution 34 containing the lignin blocking agent is extracted from the washing water tank 13 by adjusting the opening degree of the control valve V23, is pumped by the pump P2, and is supplied from the washing water tank 13 to the press filtration apparatus 32. .

Further, the washing water tank 13 is provided with a heat insulation facility 36 around it. Thereby, the aqueous solution 34 containing the lignin blocking agent in the washing water tank 13 is kept at a predetermined temperature. The cleaning water tank 13 is connected to the circulation line L11 and the cleaning liquid supply line L24. The aqueous solution 34 containing the lignin blocking agent is supplied to the cleaning water tank 13 through the cleaning liquid supply line L24. The supply amount of the aqueous solution 34 containing the lignin blocking agent is adjusted by adjusting the opening degree of the control valve V24.

The cleaning liquid obtained by the press filtration apparatus 32 is supplied to the filtrate recovery tank 14 through the sugar liquid supply line L21, and is mixed with the sugar liquid in the filtrate recovery tank 14. Further, the cleaning liquid obtained by the press filtration apparatus 32 may be circulated to the cleaning water tank 13 through the branch line L22. At this time, the opening degree of the control valves V21 and V25 is adjusted so that the control valve V21 is closed and the control valve V25 is opened.

The filtrate collection tank 14 is a tank for storing a sugar solution, a washing solution, or a mixed solution 41 in which these are mixed. In the filtrate collection tank 14, a sugar solution supply line L 31 that supplies the solution in the filtrate collection tank 14 to the ultrafiltration membrane device 15, and a filament shape that supplies the solution in the filtrate collection tank 14 to the stirring tank 21. A saccharification enzyme recovery line L11 mixed with the fungus-derived cellulase 26 is connected.

The mixed solution 41 is supplied to the ultrafiltration membrane device 15 through the sugar solution supply line L31. The sugar solution supply line L31 is provided with a control valve V31, and the supply amount of the mixed solution 41 to the ultrafiltration membrane device 15 is adjusted by adjusting the opening degree of the control valve V31. The mixed solution 41 is pumped by the pump P3 and supplied to the ultrafiltration membrane device 15.

The ultrafiltration membrane device 15 filters the mixed solution 41 and separates it into a permeate 42 and a non-permeate 43. The permeate 42 that has passed through the ultrafiltration membrane in the ultrafiltration membrane device 15 is recovered as a sugar solution. The non-permeated liquid 43 that does not pass through the ultrafiltration membrane is supplied to the filtrate collection tank 14 after the mixed solution 41 in the filtrate collection tank 14 is discharged.

The non-permeated liquid 43 recovered in the filtrate recovery tank 14 is mixed with the filamentous fungus-derived cellulase supplied to the stirring tank 21 through the saccharifying enzyme recovery line L11 and supplied to the stirring tank 21. The saccharification enzyme recovery line L11 is provided with a control valve V32, and the supply amount of the non-permeated liquid is adjusted by adjusting the opening degree of the control valve V32. Since the non-permeated liquid 43 collected in the filtrate collection tank 14 contains filamentous fungus-derived cellulase, it can be reused as the filamentous fungus-derived cellulase in the stirring tank 21.

The apparatus for producing a sugar solution using the method for producing a sugar solution according to the present invention recovers and reuses the filamentous fungus-derived cellulase adhering to the saccharification residue, thereby further reducing the amount of fungus-derived cellulase used. Can be manufactured. Thereby, the manufacturing cost of the sugar solution can be greatly reduced.

<Application of sugar solution>
The sugar solution obtained by the present invention can be used for various uses such as food raw materials, pharmaceutical raw materials, and fermentation raw materials such as chemicals. The sugar solution obtained by the present invention is used as a fermentation raw material, and various chemicals can be produced by growing microorganisms having the ability to produce chemical products. Note that growing the microorganism means that the sugar component or amino source contained in the sugar solution is used as a nutrient of 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. Examples of chemicals that can be produced by microorganisms include alcohols such as ethanol, propanol, butanol, 1,3-propanediol, 1,4-butanediol, and glycerol, acetic acid, lactic acid, pyruvic acid, succinic acid, malic acid, and itacone. Examples thereof include organic acids such as acids and citric acid, nucleosides such as inosine and guanosine, nucleotides such as inosinic acid and guanylic acid, and amine compounds such as cadaverine. Furthermore, the sugar solution obtained by the method for producing a 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 method for producing a sugar solution of the present invention will be specifically described with reference to examples in order to explain in more detail. However, the present invention is not limited to these.

(Reference Example 1) Preparation of filamentous fungus-derived cellulase (culture solution) Filamentous fungus-derived cellulase (culture solution) was prepared by the following method.

(Pre-culture)
Corn steep liquor 5% (w / vol), glucose 2% (w / vol), ammonium tartrate 0.37% (w / vol), ammonium sulfate 0.14 (w / vol), potassium dihydrogen phosphate 0.2 % (W / vol), calcium chloride dihydrate 0.03% (w / vol), magnesium sulfate heptahydrate 0.03% (w / vol), zinc chloride 0.02% (w / vol) , Iron (III) chloride hexahydrate 0.01% (w / vol), copper (II) sulfate pentahydrate 0.004% (w / vol), manganese chloride tetrahydrate 0.0008% ( w / vol), boric acid 0.0006% (w / vol), and hexamolybdate hexaammonium tetrahydrate 0.0026% (w / vol) were added to distilled water. 500mL baffle with 100mL distilled water containing The flask was placed in an Erlenmeyer flask and sterilized by autoclave at a temperature of 121 ° C. for 15 minutes. After standing to cool, separately from this, PE-M and Tween 80, which were autoclaved at 121 ° C. for 15 minutes, were respectively added to the above 500 mL baffled Erlenmeyer flasks at 0.01% (w / vol). The pre-culture medium was inoculated with Trichoderma reesei ATCC 66589 at 1 × 10 ⁵ cells / mL, and a shaking device (BIO-SHAKER BR-40LF manufactured by TAITEC) was used at a temperature of 28 ° C. for 72 hours. Pre-culture was performed by shaking culture at 180 rpm for a period of time.

(Main culture)
Corn steep liquor 5% (w / vol), glucose 2% (w / vol), cellulose (Avicel) 10% (w / vol), ammonium tartrate 0.37% (w / vol), ammonium sulfate 0.14% ( w / vol), potassium dihydrogen phosphate 0.2% (w / vol), calcium chloride dihydrate 0.03% (w / vol), magnesium sulfate heptahydrate 0.03% (w / vol) ), Zinc chloride 0.02% (w / vol), iron (III) chloride hexahydrate 0.01% (w / vol), copper (II) sulfate pentahydrate 0.004% (w / vol) ), Manganese chloride tetrahydrate 0.0008% (w / vol), boric acid 0.0006% (w / vol), and hexamolybdate hexaammonium tetrahydrate 0.0026% (w / vol) Add to distilled water so that each of the above components Was added to a 5 L stirring jar (DPC-2A, manufactured by Able) container, and autoclaved at a temperature of 121 ° C. for 15 minutes. Separately from this, 0.1% each of PE-M and Tween 80 which were autoclaved at 121 ° C. for 15 minutes, respectively, was added, and Trichoderma reesei ATCC 66589 pre-cultured in a liquid medium by the above-described method was added. 250 mL was inoculated. Thereafter, using a shaking device (BIO-SHAKER BR-40LF manufactured by TAITEC), shaking culture was performed at 28 ° C. for 87 hours under the conditions of 300 rpm and aeration volume of 1 vvm. Then, after centrifuging, the supernatant was subjected to membrane filtration (Stellicup-GV, material: PVDF, manufactured by Millipore). The obtained culture broth was used as a filamentous fungus-derived cellulase in the following examples.

In the following examples and comparative examples, the sugar concentration and the activity of cellulase derived from filamentous fungi were measured as follows.

Reference Example 2 Measurement of Sugar Concentration Each concentration of glucose and xylose contained in the sugar solution was quantified by comparison with a standard under the following high performance liquid chromatography (HPLC) conditions.
(HPLC conditions)
Column: Luna NH ₂ (Phenomenex)
Mobile phase: Milli Q: Acetonitrile = 25: 75
Flow rate: 0.6 mL / min Reaction liquid: None Detection method: RI (differential refractive index)
Temperature: 30 ° C.

(Reference Example 3) Measurement of activity of filamentous fungus-derived cellulase The enzymatic activity of filamentous fungus-derived cellulase is divided into two types of degradation activity: (1) Avicel degradation activity and (2) Xylan degradation activity. Was evaluated.

(1) Avicel decomposition activity To the recovered enzyme solution (100 μL), 1 g / L of Avicel (Merck) and sodium acetate buffer (pH 5.0) were added to a concentration of 100 mM, and the temperature was 50 ° C. The reaction was performed for 24 hours. The reaction solution was adjusted with a 1 mL tube, and the reaction was performed while rotating and mixing under the above conditions. After the reaction, the tube was centrifuged, and the glucose concentration of the supernatant component was measured. The glucose concentration was measured according to the method described in Reference Example 2. For the Avicel degradation activity, the generated glucose concentration (g / L) was used as the activity value as it was.

(2) Xylan decomposition activity To the enzyme solution, 10 g / L of xylan (Birch wood xylan, manufactured by Wako Pure Chemical Industries, Ltd.) and sodium acetate buffer (pH 5.0) were added to a concentration of 100 mM at 50 ° C. The reaction was performed for 4 hours. The reaction solution was adjusted with a 1 mL tube, and the reaction was performed while rotating and mixing under the above conditions. After the reaction, the tube was centrifuged, and the xylose concentration of the supernatant component was measured. The xylose concentration was measured according to the method described in Reference Example 2. For the xylose decomposition activity, the generated xylose concentration (g / L) was directly used as the activity value.

<Example 1: Hydrolysis of cellulose-containing biomass (step (1))>
[Pretreatment of cellulose-containing biomass]
(Ammonia treatment of cellulose-containing biomass (Pretreatment 1))
Rice straw was used as the cellulose-containing biomass. The rice straw was put into a small reactor (manufactured by pressure-resistant glass industry, TVS-N2 30 ml) and cooled with liquid nitrogen. Ammonia gas was flowed into the reactor, and the sample was completely immersed in liquid ammonia. The reactor lid was closed and left at room temperature for about 15 minutes. Subsequently, it processed in the 150 degreeC oil bath for 1 hour. After the treatment, the reactor was taken out from the oil bath, and immediately after ammonia gas leaked in the fume hood, the reactor was further evacuated to 10 Pa and dried. This was used as the pre-processed product 1 in the following examples.

(Hydrothermal treatment of cellulose-containing biomass (Pretreatment 2))
Rice straw was used as the cellulose-containing biomass. The rice straw was soaked in water and autoclaved (manufactured by Nitto Koatsu Co., Ltd.) for 20 minutes at 180 ° C. with stirring. The pressure at that time was 10 MPa. After the treatment, the solution component (hereinafter referred to as hydrothermal treatment liquid) and the treated biomass component were subjected to solid-liquid separation using centrifugation (3000G). This treated biomass component was used as a pretreated product 2 in the following examples.

[Hydrolysis of cellulose-containing biomass by cellulase derived from filamentous fungi]
The aforementioned pretreated product 1 and pretreated product 2 were each hydrolyzed with filamentous fungus-derived cellulase. Distilled water was added to each pretreated product (0.5 g), and then 0.5 mL of the filamentous fungus-derived cellulase prepared in Reference Example 2 was added. Thereafter, distilled water was further added to each pretreated product (0.5 g) so that the total weight was 10 g. Further, dilute sulfuric acid or dilute caustic soda was added to adjust the pH of each of the present compositions to be in the range of 4.5 to 5.3. Each of the present compositions was transferred to a branched reaction vessel (manufactured by Tokyo Rika Kikai Co., Ltd., φ30, NS14 / 23). Thereafter, the branched reaction vessel was placed in a thermostatic bath (MG-2200, manufactured by Tokyo Rika Co., Ltd.), and the present composition was used at 50 ° C. for 24 hours using a small stirrer (CPS-1000, manufactured by Tokyo Rika Co., Ltd.). The mixture was hydrolyzed while keeping it warm and stirring. The hydrolyzate obtained from the pretreatment product 1 is referred to as “hydrolyzate 1”, and the hydrolyzate obtained from the pretreatment product 2 is referred to as “hydrolyzate 2” in the following steps (2) and subsequent examples. did.

<Example 2: Solid-liquid separation of hydrolyzate (step (2))>
The hydrolyzate 1 and hydrolyzate 2 obtained in Example 1 were subjected to solid-liquid separation by centrifugation (3000 G, 10 minutes), respectively, and separated into a sugar solution (6 g) and a saccharification residue (4 g). The sugar solution and saccharification residue obtained from the hydrolyzate 1 are designated as “sugar solution 1” and “saccharification residue 1”, and the sugar solution and the saccharification residue obtained from the hydrolyzate 2 are designated as “sugar solution 2” and “ As saccharification residue 2 ", it was used in the examples after step (3). Further, the sugar concentrations (glucose and xylose concentrations) of the obtained sugar solution 1 and sugar solution 2 were measured by the method described in Reference Example 1. Table 1 shows the measurement results of the sugar concentrations (glucose and xylose concentrations) of the obtained sugar solution 1 and sugar solution 2.

<Example 3: Washing of saccharification residue (step (3)) and filtration of the obtained washed solution (step (4))>
Each saccharification residue obtained in Example 2 was washed with an aqueous solution containing a lignin blocking agent at room temperature. As lignin blocking agents, BSA (bovine serum-derived albumin, manufactured by Sigma-Aldrich), casein (manufactured by Sigma-Aldrich), DDGS (maize distillation residue, BP-50, manufactured by Wilbur-Ellis), CSL (corn steep liquor, prince Cornstarch Co., Ltd.), skim milk (Wako Pure Chemical Industries, Ltd.), and peptone (BBL Peptone, Becton Dickinson) were used. A lignin blocking agent was added to sterilized water to a final concentration of 5 g / L to prepare a washed solution. 6 g (6 mL) of these washed solutions were added to “saccharification residue 1” and “saccharification residue 2” and mixed. After mixing, the mixture was allowed to stand at room temperature for 30 minutes, and then centrifuged (3000 G, 10 minutes) for solid-liquid separation, and 6 g of each washed solution of saccharification residue 1 and saccharification residue 2 was collected as each centrifugation supernatant. The collected supernatant component was subjected to microfiltration using a Milex HV filter unit (33 mm, PVDF, pore diameter 0.45 μm). The obtained filtrate was filtered through an ultrafiltration membrane having a fractional molecular weight of 10,000 (VARISPIN 20 material: PES, manufactured by Sartorius steady biotech), and centrifuged at 4500 G until the membrane fraction became 1 mL. Further, 6 mL of distilled water was added to the membrane fraction, and centrifuged at 4500 G until the membrane fraction reached 1 mL again. Thereafter, the enzyme was recovered from the membrane fraction. Each activity of the recovered enzyme was measured according to Reference Example 3. For comparison with the prior art, as Comparative Example 1, the activity of each recovered enzyme component when the saccharification residue was washed only with distilled water (not including the lignin blocking agent) was used as a reference value, and the recovered enzyme as a relative value. The activity of the components is shown in Tables 2 and 3.

As is apparent from Tables 2 and 3, it was found that washing with an aqueous solution containing a lignin blocking agent increased the recovery of cellulase derived from filamentous fungi, and the Avicel degradation activity in the recovered enzyme component was particularly increased.

<Example 4: Filtration of a mixed solution of the sugar solution of step (2) and the washed solution (step (4))>
The sugar solution 1 (6 g) of Example 2 and the washed solution (6 g) of Example 3 (washed solution obtained by washing saccharification residue 1 with CSL) were mixed and filtered through an ultrafiltration membrane. Went. Microfiltration was performed using a Milex HV filter unit (33 mm, made by PVDF, pore diameter 0.45 μm). The obtained filtrate was filtered through an ultrafiltration membrane having a fractional molecular weight of 10,000 (VARISPIN 20 material: PES, manufactured by Sartorius Steadim Biotech), and centrifuged at 4500 G until the membrane fraction became 1 mL. Further, 6 mL of distilled water was added to the membrane fraction, and centrifuged at 4500 G until the membrane fraction reached 1 mL again. Thereafter, the enzyme was recovered from the membrane fraction. Each activity of the recovered enzyme was measured according to Reference Example 3. In addition, for comparison of the recovered enzyme activity, after mixing the sugar solution of step (2) with the washed solution of the saccharification residue of Comparative Example 1, it is filtered through an ultrafiltration membrane to obtain a non-permeate solution. The recovered enzyme activity obtained in the step of recovering the filamentous fungus-derived cellulase (Comparative Example 2) is shown in Table 4 as relative values based on the reference (1).

As is clear from Table 4, it was found that in Example 4, each recovered enzyme activity increased compared to Comparative Example 2. Moreover, in Example 4, it turned out that the difference of each collection | recovery enzyme activity relative to Comparative Example 2 is large with respect to Example 3.

<Example 5: Effect of temperature during washing of saccharification residue in aqueous solution containing lignin blocking agent>
When washing the saccharification residue using an aqueous solution containing a lignin blocking agent, the effect of the temperature during washing on the saccharification residue was examined. What added the washing | cleaning solution to the saccharification residue 1 was immersed in the hot water bath set to each temperature of 40 degreeC, 50 degreeC, and 60 degreeC, and it was left to stand for 30 minutes. Thereafter, the recovered enzyme was obtained in the same manner as in Example 3, and each enzyme activity was measured.

As shown in Tables 5 and 6, it was found that the enzyme activity of the washed solution recovered by washing saccharification residue 1 in the temperature range of 40 to 60 ° C. was increased as compared to room temperature.

<Example 6: Washing of saccharification residue with aqueous solution containing lignin blocking agent and ammonium sulfate>
In washing saccharification residue with an aqueous solution containing a lignin blocking agent, the washing effect in the case of adding ammonium sulfate as an inorganic salt was examined. As lignin blocking agents, BSA (bovine serum-derived albumin, manufactured by Sigma Aldrich) and CSL (Corn Steep liquor, manufactured by Oji Cornstarch Co., Ltd.) were used. The lignin blocking agent was added to sterilized water to a final concentration of 5 g / L, and in this example, ammonium sulfate was further added to 1 g / L, 5 g / L, and 10 g / L to obtain a washed solution. Was prepared. 6 g (6 mL) of these washed solutions was added to “saccharification residue 1” and mixed. After mixing, the mixture was allowed to stand at room temperature for 30 minutes, and then centrifuged (3000 G, 10 minutes) for solid-liquid separation, and 6 g of the washed solution of saccharification residue 1 was collected as each centrifugal supernatant. The collected supernatant components were measured for each enzyme activity in the same procedure as in Example 3. The results are shown in Table 7 and Table 8.

As is apparent from Tables 7 and 8, it was found that the enzyme activity was increased by adding ammonium sulfate to the further washed solution. In particular, this effect has been found to be great for xylan decomposition activity.

<Example 7: Production of lactic acid>
Using the sugar solution 1 and the sugar solution 2 of Example 2 as fermentation raw materials, the Lactococcus lactis JCM7638 strain was statically cultured at a temperature of 37 ° C. for 24 hours. The concentration of L-lactic acid contained in the culture solution was analyzed under the following conditions. The results are shown in Table 9.
Column: Shim-Pack SPR-H (manufactured by Shimadzu Corporation)
Mobile phase: 5 mM p-toluenesulfonic acid (flow rate 0.8 mL / min)
Reaction solution: 5 mM p-toluenesulfonic acid, 20 mM Bistris, 0.1 mM EDTA · 2Na (flow rate 0.8 mL / min)
Detection method: Electrical conductivity Temperature: 45 ° C

As shown in Table 9, it was confirmed that L-lactic acid can be produced by using sugar solution 1 and sugar solution 2 as fermentation raw materials.

DESCRIPTION OF SYMBOLS 10 Sugar liquid production apparatus 11 Hydrolysis reaction tank 12 Solid-liquid separation apparatus 13 Washing water tank 14 Filtrate collection tank 15 Ultrafiltration membrane apparatus 21 Stirring tank 22 Cellulose containing biomass 23 Stirring apparatus 24, 36 Thermal insulation equipment 25, 31 Supply port 26 Filamentous cellulase 28 Hydrolyzate 32 Press filtration apparatus 33 Compressor 34 Aqueous solution containing lignin blocking agent 35 Water inlet 41 Mixed solution 42 Sugar solution (permeate)
43 Non-permeate L11 Saccharification enzyme recovery line

Claims (10)

1. A method for producing a sugar solution from cellulose-containing biomass,
   Step (1): A step of hydrolyzing the cellulose-containing biomass with a filamentous fungus-derived cellulase to obtain a hydrolyzate,
   Step (2): Solid-liquid separation of the hydrolyzate into a sugar solution and a saccharification residue,
   Step (3): washing the saccharification residue with an aqueous solution containing a lignin blocking agent to obtain a washed solution containing the filamentous fungus-derived cellulase contained in the saccharification residue, and step (4): the washing Filtering the solution containing the solution, and recovering the non-permeate containing the filamentous fungus-derived cellulase,
   A method for producing a sugar solution, comprising:
2. The lignin blocking agent is corn steep liquor, peptone, yeast extract, meat extract, casein, skim milk, bovine serum-derived albumin, ethanol fermentation distillation residue, zein, fish processing waste, meat processing waste, whey protein, grain processing waste The manufacturing method of the sugar liquid of Claim 1 which is 1 or more selected from the group which consists of a thing, sugar processing waste, food, algal protein, soybean protein, bacterial protein, and fungal protein.
3. The method for producing a sugar solution according to claim 1 or 2, wherein in the step (4), the filtration of the washed solution is an ultrafiltration membrane.
4. Mixing the sugar solution obtained in step (2) with the washed solution obtained in step (3),
   The sugar according to any one of claims 1 to 3, wherein in the step (4), a mixed solution containing the sugar solution and the washed solution is filtered to collect a permeate containing the sugar solution. Liquid manufacturing method.
5. The method for producing a sugar liquid according to any one of claims 1 to 4, wherein in step (3), the temperature of the aqueous solution containing the lignin blocking agent is 40 to 60 ° C.
6. The method for producing a sugar liquid according to any one of claims 1 to 5, wherein the aqueous solution containing the lignin blocking agent contains an inorganic salt.
7. The method for producing a sugar solution according to any one of claims 1 to 6, wherein the filamentous fungus-derived cellulase is derived from a Trichoderma microorganism.
8. The method for producing a sugar liquid according to any one of claims 1 to 7, wherein the non-permeating liquid is mixed with the filamentous fungus-derived cellulase in the step (1).
9. The method for producing a sugar liquid according to any one of claims 1 to 8, wherein in the step (2), the solid-liquid separation of the hydrolyzate is performed by press filtration.
10. A step of producing a sugar solution by the method for producing a sugar solution according to any one of claims 1 to 9,
    Culturing a microorganism having the ability to produce a chemical using the sugar solution as a fermentation raw material;
    A method for producing a chemical product.

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