JP6474150B2 - Saccharification method of biomass raw material - Google Patents

Description

  The present invention relates to a biomass raw material saccharification method in which holocelluloses in a biomass raw material in which lignins coexist are saccharified using a cellulose hydrolase.

  In recent years, as a means for solving energy and environmental problems, there is a strong demand for the development of technology that effectively uses renewable materials. One of such technologies is biorefinery technology, and attention is focused on technology that efficiently uses biomass resources and efficiently converts it into chemicals and fuels. In particular, there is a strong demand for the development of technology that uses holocelluloses, which are the most abundant biomass resources in nature.

  When converting holocelluloses into useful substances such as chemicals and fuels, it is necessary to decompose the holocelluloses and convert (saccharify) them into low molecular weight holocellulose derivatives such as sugars. As a method for decomposing holocelluloses, a method of hydrolyzing holocelluloses with acid or alkali, a method of decomposing by heat, a method of irradiating with microwaves, a method of supercritical water treatment, laser heating, etc. may be used alone or A method of using them in combination is known. However, in practice, these methods are only limitedly used because of complicated operations and expensive equipment. On the other hand, a biological method using cellulose hydrolase and the like is the most popular method as a means for saccharification of holocelluloses because it is easy to operate and easy to manage.

  When biomass saccharification is performed using a cellulose hydrolase such as cellulase, the biomass raw material is previously pulverized, steamed, pyrolyzed, pressure cracked, electron beam, γ-ray, Those subjected to microwave treatment, acid treatment, alkali treatment and the like are used.

Polymer anionic surface activity with (meth) acrylic acid ester and (meth) acrylic acid (salt) as essential constituent monomers in biomass raw material for the purpose of enhancing the action of cellulose hydrolase on holocelluloses A method (Patent Document 1) in which an agent is added and saccharification of biomass by a hydrolase is disclosed. However, Patent Document 1 does not disclose the use of poly (meth) acrylic acid (salt) in the enzymatic saccharification reaction.
Further, it is disclosed that a biomass raw material is pulverized in the presence of a basic compound and an anionic polymer for the purpose of promoting lignin peeling from cellulose and enhancing the action of an enzyme on cellulose (Patent Document 2). ). Patent Document 2 does not disclose enzymatically saccharifying cellulose by adding a polyacrylate after pulverization.

  On the other hand, when lignins coexist in the pretreated biomass material, the lignins strongly adsorb cellulose hydrolase, and this phenomenon causes degradation of holocelluloses by saccharification (saccharification). It is known that the efficiency of the reaction is significantly suppressed. That is, it has been found that one of the important points for efficiently decomposing holocelluloses in biomass raw materials is to control the adsorption of cellulose hydrolase to lignins. Further, when cellulose hydrolase is adsorbed on lignins, it is usually difficult to reuse the cellulose hydrolase. In the industry, cost reduction of cellulose hydrolase is important, and it is also desired to reuse cellulose hydrolase.

  When saccharifying holocelluloses in biomass raw materials containing lignin with enzymes, as a method of avoiding the effects of lignins in biomass raw materials, before mixing the enzymes, for example, alkali cooking, kraft cooking, ozone A method for treating a biomass raw material by treatment, hydrogen peroxide treatment, etc. (Patent Document 3), and a method for adding a skim milk, which is a substance that inhibits the adsorption of hydrolase to lignin (Patent Document 3) Document 4), a method of saccharifying a biomass raw material after adding a lignin derivative that is a reaction product of lignins and a hydrophilic compound (Patent Document 5), and the like are known.

  However, the saccharification methods of Patent Documents 3 to 5 are not satisfactory in terms of saccharification efficiency and productivity. Moreover, even if these methods improve saccharification efficiency and productivity, the operation is complicated, the apparatus is expensive, and there are also problems in safety and the like. There is a need for a method that can avoid the influence of lignins by a simpler operation and efficiently saccharify biomass raw materials.

JP 2012-75378 A JP 2014-117207 A JP 2008-92910 A JP 2009-72144 A International Publication 2011/111664 Pamphlet

  An object of the present invention is to provide a simple method for saccharification of biomass material that can efficiently utilize cellulose hydrolase at the same time as saccharifying the holocelluloses in the biomass material efficiently with cellulose hydrolase. . Furthermore, the subject of this invention is providing the manufacturing method of bioethanol using the saccharification method of biomass raw material.

  As a result of intensive studies in view of the above problems, the present inventors have focused on the fact that poly (meth) acrylic acid (salt) acts as a substance that can inhibit the adsorption of lignins and cellulose hydrolase, A method in which poly (meth) acrylic acid (salt) is added in advance to at least one of a biomass raw material and a cellulose hydrolase coexisting with lignins, and then the biomass raw material and the cellulose hydrolase are mixed to saccharify the biomass raw material. The present invention has been completed.

  Furthermore, the present inventors have used the above saccharification method to saccharify a biomass raw material using poly (meth) acrylic acid (salt), or at the same time as saccharification, by subjecting saccharides to alcohol fermentation by microorganisms such as yeast to produce ethanol. As a result, the present invention was completed.

That is, this invention consists of the following.
1. A method for saccharification of holocelluloses in a biomass raw material in which lignins coexist, including the following steps (1) and (2):
Step (1): A step of adding a poly (meth) acrylic acid (salt) in advance to at least one of the biomass raw material and the cellulose hydrolase to obtain a mixed solution,
Step (2): By mixing the mixture obtained in step (1) with cellulose hydrolase or biomass raw material, the biomass raw material and cellulose hydrolase are reacted, and the holocelluloses in the biomass raw material are converted into enzymes. The process of saccharification.
2. The method for producing ethanol according to the saccharification method described in the preceding item 1, comprising subjecting the saccharide to alcohol fermentation after the enzyme saccharification step of step (2) or simultaneously with the enzyme saccharification step.

The saccharification method of holocelluloses in biomass raw materials in which the lignins of the present invention coexist is one in which adsorption of cellulose hydrolase to lignins is remarkably suppressed, and saccharification of holocelluloses by cellulose hydrolase is suppressed. Without hindering, the holocelluloses in the biomass raw material can be efficiently saccharified.
In addition, the cellulose hydrolase after the saccharification reaction can be reused, and the cellulose hydrolase can be used efficiently. Since the method for saccharification of biomass raw material of the present invention does not require special treatment such as decomposition and removal of lignins, it is very efficient in terms of energy, time, and practical operation. Saccharification of holocelluloses becomes possible.
In addition, the saccharide obtained by the saccharification method of the present invention can be used for alcohol fermentation, lactic acid fermentation and the like, and can produce alcohol and lactic acid such as ethanol and the like by using biomass raw material with high efficiency.

  Hereinafter, the present invention will be described in more detail. The present invention is a method for saccharifying holocelluloses in a biomass raw material in which lignins coexist, including at least steps (1) and (2). In the saccharification method of the present invention, irreversible adsorption of lignin and cellulose hydrolase is suppressed by poly (meth) acrylic acid (salt), and an enzymatic saccharification reaction of biomass raw material is performed by cellulose hydrolase. . In the saccharification method of the present invention, since the cellulose hydrolase is used for saccharification of holocelluloses without being deprived of lignins, the holocelluloses can be efficiently saccharified.

<Process (1)>
Step (1) is a step in which poly (meth) acrylic acid (salt) is added in advance to at least one of the biomass raw material and cellulose hydrolase to obtain a mixed solution.

  There is no restriction | limiting in particular in the kind of biomass raw material which can be used by this invention, What kind of thing may be used as long as lignins coexist and holocelluloses are contained. Examples of biomass raw materials include woody materials (tree trunks, branches, roots, leaves of trees, etc. of conifers and broadleaf trees, waste materials, pulps, papers, etc.), herbaceous materials (rice, wheat, corn, Examples include sugarcane, beans, and other crops, weed stems and roots, mowing grass, and the like, and residues thereof. One or more of these can be used. In particular, woody materials derived from conifers (cedar, larch, cypress, etc.) and woody materials derived from broadleaf trees (napa palm, oil palm, kunugi, etc.), which are lignocellulosic biomass materials rich in lignins Can be used.

  Prior to being used in step (1), the biomass raw material is preferably subjected to physical treatment such as cutting treatment or pulverization treatment so that the saccharification reaction proceeds efficiently, and is made into an appropriate shape and size. In order to perform physical processing such as cutting and pulverization of the biomass material more appropriately, the biomass material is in a dry state. The biomass raw material is dried by, for example, a normal wind drying method, and is preferably dried to a moisture content of 20% by mass or less.

When the biomass raw material is cut, the size of the equipment for the cutting treatment and the biomass raw material after the cutting treatment is not particularly limited, but for example, 5 cm or less, preferably 2 cm or less, particularly preferably with a rotary cutter or the like. May be a chip piece of 1 cm or less.
When the biomass raw material is pulverized, the apparatus for pulverization and the size after the pulverization are not particularly limited. For example, the average particle size of the biomass raw material is 500 μm or less, preferably 200 μm or less, using a high-pressure compression roll mill or the like. More preferably, the thickness may be 100 μm or less. An average particle diameter can be measured by the method as described in the Example mentioned later.

The temperature at the time of cutting and pulverization is not particularly limited and may be in the range of −100 to 200 ° C., but 2 to 100 ° C. is preferable in order not to consume extra energy.
The time for cutting and pulverization varies depending on the equipment to be used, the size of the biomass raw material after cutting and pulverization, the state of drying of the biomass raw material, etc., but is preferably within 30 minutes from the viewpoint of economy.

  The biomass raw material after the cutting and pulverization treatment may be subjected to the step (1) as it is, or may be further pretreated. Examples of the pretreatment include steaming treatment, blasting treatment, hydrothermal treatment, acid (sulfuric acid, dilute sulfuric acid, etc.) treatment or alkali treatment, etc., and by these treatments, the reactivity of cellulose hydrolase to holocelluloses is increased. Can be improved. Further, as a pretreatment, a treatment such as mixing with a peroxide in an alkaline aqueous solution may be performed.

  The holocelluloses contained in the biomass raw material mean polysaccharides containing a lot of β-1,4 glucoside bonds. Specific examples of holocelluloses include cellulose, hemicellulose, and modified products thereof. Moreover, the biomass raw material has not been subjected to lignin removal or decomposition treatment, and contains lignin. Lignins include lignin or modified products thereof.

As the cellulose hydrolase used in the present invention, any cellulose hydrolase capable of hydrolyzing holocelluloses can be used without limitation. Cellulose hydrolase derived from animals, plants, and microorganisms can be used alone, or Can be used as a mixture. As the cellulose hydrolase, cellulase, hemicellulase and the like are often used from the viewpoint of saccharification efficiency. Cellulase is an enzyme that hydrolyzes the glucoside bond of β1,4 glucan of cellulose. Cellulase (EC number 3.2.1.4), exo-1,4β-D-glucosidase (EC number) 3.2.74) and exocellobiohydrolase (EC number 2.2.1.91) can be used.
More specifically, cellulases include “Cellulase“ Onoka ”3S, Cellulase Y-NC, etc.” (manufactured by Yakult Yakuhin Co., Ltd.), “Sucrase C, etc.” (manufactured by Mitsubishi Chemical Foods), “Cellulase A” "Amano" 3, Cellulase T "Amano" 4 etc. "(Amano Enzyme Co., Ltd.)," TP-60, Mecelase "(Meiji Seika Co., Ltd.)," Cellulosin AC40 "," Cellulosin AL "," Cellulosin T3 " Etc. (manufactured by HIBI), “Entilon CM, MHC, Biohit, Biostar, etc.” (manufactured by Nitto Kasei Kogyo Co., Ltd.), “Celcrust 1.5L”, “CellicCTec2,” “CellicHTec2,” “Ultra Flo L”, “Novazyme 188”, “Phenozyme”, “Viscozyme”, etc. (manufactured by Novozyme Japan), “Accel” erase "it is possible to use the (Genencor Co., Ltd.), and the like.

  In the step (1), a high molecular weight substance is used as an additive in order to efficiently perform a saccharification reaction. As a high molecular weight body, it is necessary to have a function which suppresses adsorption | suction of lignin and a cellulose hydrolase. In the present invention, poly (meth) acrylic acid (salt) is used as a high molecular weight body having such a function.

Poly (meth) acrylic acid (salt), which is a high molecular weight substance used in the present invention, is obtained by solution polymerization, emulsion polymerization, suspension of (meth) acrylic acid (salt) monomer (hereinafter referred to as “MA”). It can be obtained by polymerization according to a known polymerization method such as turbid polymerization.
In the present invention, (meth) acrylic acid represents methacrylic acid and / or acrylic acid, and (meth) acrylic acid (salt) represents (meth) acrylic acid and / or a salt of (meth) acrylic acid. Represent. In addition, the (meth) acrylic acid ester is not contained in the monomer which comprises poly (meth) acrylic acid (salt).
In addition, preferable poly (meth) acrylic acid (salt) of the present invention can be exemplified by polyacrylic acid sodium salt, polyacrylic acid ammonium salt, polymethacrylic acid sodium salt, and polymethacrylic acid ammonium salt.
As a specific polymerization method for obtaining a high molecular weight product, for example, in the case of solution polymerization, the monomer composition is dissolved in an organic solvent such as lower alcohol, and the reaction is performed under an inert gas atmosphere such as nitrogen or argon. Examples thereof include a method of obtaining a high molecular weight body by adding a radical polymerization initiator such as an oxide or an azo compound, followed by heating and stirring. The form of the high molecular weight obtained may be either powder or solution, but is preferably a transparent solution when dissolved in water. Moreover, when refine | purifying a high molecular weight body, it can carry out by general purification methods, such as a reprecipitation method, a dialysis method, and an ultrafiltration method. When (meth) acrylic acid is used as a monomer for obtaining a high molecular weight product, it is neutralized with sodium hydroxide or aqueous ammonia after polymerization to obtain poly (meth) acrylic acid (salt). Can do.

  Moreover, the high molecular weight body used for this invention can also utilize a commercial item simply. Examples of commercially available products include the following product names. For example, “Aquaric series” (Aquaric is a registered trademark of Sanyo Kasei Co., Ltd.), “Aron series”, “Aronbis series”, “Jurimer series” (Aron, Alonbis, and Jurimer are from Toagosei Co., Ltd.) Registered trademark), “viscomate series” (viscomate is a registered trademark of Toagosei Co., Ltd.), and the like. When other commercially available poly (meth) acrylic acid is used, it is preferable to use poly (meth) acrylic acid (salt) neutralized with sodium hydroxide or aqueous ammonia.

  The high molecular weight body of the present invention preferably has an average molecular weight of 2,500 or more, and preferably 10,000,000 or less from the viewpoint of ease of handling. More preferably, it is 5,000 or more and 1,000,000 or less, and more preferably about 500,000. In the present invention, the average molecular weight means a mass average molecular weight. The mass average molecular weight can be measured by gel permeation chromatography using polyethylene glycol as a standard sample.

  The addition amount of the high molecular weight body depends on the amount of lignin coexisting in the biomass material, but is 0.01 to 50% by mass, preferably 0.1 to 20% by mass, based on the dry mass of the biomass material. Preferably it is 1-10 mass%. If the amount of the high molecular weight is too small, the influence of lignin on cellulose hydrolase cannot be avoided.

  There is no particular limitation on the method for adding the high molecular weight material as long as it is before mixing the biomass raw material and the cellulose hydrolase, and either batch charging or split charging may be used. Moreover, a high molecular weight body may be previously mixed with a biomass raw material, and may be previously mixed with a cellulose hydrolase. When the biomass material and the high molecular weight material are mixed in advance, it is preferable to mix the high molecular weight material after pulverization and cutting of the biomass raw material. Thereby, the masking effect of the lignin or cellulose hydrolase by the high molecular weight substance is sufficiently exhibited. In addition, from the viewpoint of recovering the activity of cellulose hydrolase, it is desirable to previously mix cellulose hydrolase and a high molecular weight substance. Moreover, in order to disperse | distribute a high molecular weight body uniformly, it is good to mix, stirring. Solvents necessary for the saccharification reaction, such as water and alcohol, may be added and mixed in a timely manner. In addition, nonionic surfactants such as polyoxyethylene alkyl ethers and polyoxyethylene sorbitan fatty acid esters, water-soluble polymers such as polyethylene glycol, maleic acid, etc., as components that exhibit the function of relaxing the crystal structure of holocelluloses An appropriate amount of an anionic surfactant such as a copolymer containing a salt thereof and a salt thereof may be added alone or in appropriate combination.

  The pH condition of the mixed liquid of biomass raw material and high molecular weight substance, or the mixed liquid of cellulose hydrolase and high molecular weight substance is not particularly limited, but is less than pH 7, preferably pH 4 in order to smoothly shift to the enzymatic saccharification reaction. ~ 6 is good. The pH can be adjusted using acid or alkali. Examples of the acid include sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, formic acid, acetic acid, and citric acid, and examples of the alkali include sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, and various organic amines.

The stirring and mixing of the biomass raw material and the high molecular weight substance, or the cellulose hydrolase and the high molecular weight substance is not particularly limited as long as the mixture can be uniformly dispersed. For example, a ribbon type mixer or a paddle type mixer The thing with high stirring efficiency is preferable.
The stirring and mixing time is not particularly limited, and it is sufficient that the biomass raw material and the high molecular weight material, or the cellulose hydrolase and the high molecular weight material are mixed and stirred uniformly, but about 30 minutes is sufficient. .
Furthermore, the temperature of the stirring and mixing is not particularly limited as long as the high molecular weight body can mask lignins or cellulose hydrolase. Usually, 1 to 100 ° C., and 60 ° C. or less is preferable in order not to consume extra energy.

  By performing the step (1), a mixed liquid of a biomass raw material and a high molecular weight body for an enzymatic saccharification reaction or a mixed liquid of a cellulose hydrolase and a high molecular weight body is obtained. This mixed liquid can usually be obtained as a slurry liquid.

<Process (2)>
In the step (2), the biomass liquid or the cellulose hydrolase is mixed with the mixed liquid obtained in the step (1), and the biomass raw material and the cellulose hydrolase are allowed to react, whereby the holocelluloses in the biomass raw material are reacted. Is a step of enzymatic saccharification.

  In the mixed solution obtained in the step (1), the lignin or cellulose hydrolase is masked by the high molecular weight substance. Therefore, in the step (2), when the biomass raw material and the cellulose hydrolase are mixed, the adsorption of the lignin to the cellulose hydrolase is suppressed, and the saccharification of the holocellulose in the step (2) proceeds efficiently. Moreover, since no special treatment is required to remove and decompose lignins, it is very efficient in terms of energy, time, and practical operation. Oligosaccharides such as cellobiose and cellotriose can be obtained. When the saccharide obtained by the enzymatic saccharification treatment is used for alcohol fermentation, lactic acid fermentation or the like, it is preferable to decompose it to a monosaccharide.

  The conditions for the saccharification treatment in step (2) may be any as long as the saccharification reaction can proceed smoothly. For example, water, an organic solvent, and a mixed solvent thereof can be used as the reaction solvent. As the water, tap water, industrial water, distilled water, ion-exchanged water and the like can be used, and these may be used alone or in combination. As the organic solvent, hydrocarbon solvents, ether solvents, chlorine solvents, alcohol solvents, ketone solvents, dioxane, tetrahydrofuran, acetonitrile, dimethylformamide, pyridine, etc. can be used. May be. As the organic solvent, it is preferable to use an alcohol solvent such as ethanol. The organic solvent can be used at a concentration of 10% by mass or less, preferably 5% by mass or less. Moreover, the liquid mixture of a biomass raw material and a cellulose hydrolase is pH 2-10, Preferably it is pH 3-7. An acid or alkali can be used to adjust the pH. Examples of the acid include sulfuric acid, hydrochloric acid, nitric acid, phosphoric acid, formic acid, acetic acid, and citric acid, and examples of the alkali include sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonia, and various organic amines. The reaction temperature is 10 to 90 ° C, preferably 20 to 70 ° C, more preferably 40 to 65 ° C. The reaction time is usually 30 minutes to 5 days, preferably 12 hours to 3 days.

  Ethanol can be obtained by performing alcoholic fermentation using a known method after the enzymatic saccharification step or simultaneously with the enzymatic saccharification step. Alcohol fermentation may be performed using microorganisms such as yeasts and bacteria that can ferment sugars to ethanol, and microorganisms such as yeasts and bacteria that can ferment sugars to ethanol by genetic recombination. As microorganisms capable of alcohol fermentation, ethanol-fermenting bacteria such as Saccharomyces, Zymomonas, and Pichia can be used.

When alcoholic fermentation is performed simultaneously with the enzymatic saccharification step, the pH and temperature of the reaction conditions of the enzymatic saccharification step are preferably set to conditions that allow both saccharification reaction and alcoholic fermentation. For example, the pH is preferably 4-7 and the temperature is preferably 20-40 ° C.
When the enzymatic saccharification step and the alcohol fermentation are performed separately, it is desirable that the alcohol fermentation step has conditions suitable for alcohol fermentation, and the pH is preferably 4 to 8 and the temperature is preferably 40 to 60 ° C.

  In alcohol fermentation, when the ethanol concentration in the reaction solution increases, the reaction efficiency deteriorates. Therefore, ethanol may be separated and recovered from the reaction solution as needed. For example, alcohol fermentation may be carried out while separating and recovering ethanol with an evaporator or the like. In this case, the temperature is preferably 60 ° C. or lower in order to avoid deactivation of cellulose hydrolase and microorganisms. Finally, the obtained ethanol can be recovered by distillation or the like, and re-distillation or the like may be performed to increase the purity of ethanol.

  Hereinafter, although an example and a comparative example explain the present invention still in detail, the present invention is not limited to these.

First, in Preparation Examples 1 to 6 below, preparation examples of high molecular weight materials used in the saccharification method for holocelluloses of the present invention are shown.
<Preparation Example 1>
Polyacrylic acid (average molecular weight 5,000, manufactured by Wako Pure Chemical Industries, Ltd.) was prepared. The polyacrylic acid was dissolved in ion-exchanged water and adjusted to 5% by mass while neutralizing to pH 7 with 6N and 1N sodium hydroxide solutions to obtain a high molecular weight 1 solution.
<Preparation Example 2>
A high molecular weight product 2 solution was obtained in the same manner as in Preparation Example 1 except that polyacrylic acid (average molecular weight 25,000, manufactured by Wako Pure Chemical Industries, Ltd.) was used.

<Preparation Example 3>
A high molecular weight 3 solution was obtained in the same manner as in Preparation Example 1 except that polyacrylic acid (average molecular weight 1,000,000, manufactured by Wako Pure Chemical Industries, Ltd.) was used.
<Preparation Example 4>
Polyacrylic acid (average molecular weight 1,000,000, manufactured by Wako Pure Chemical Industries, Ltd.) was prepared. The polyacrylic acid was dissolved in ion exchange water and adjusted to 5% by mass while neutralizing to pH 7 with 30% by mass and 10% by mass ammonia water to obtain a high molecular weight 4 solution.

<Preparation Example 5>
A high molecular weight solution 5 was obtained in the same manner as in Preparation Example 1 except that polymethacrylic acid (average molecular weight 100,000, manufactured by Wako Pure Chemical Industries, Ltd.) was used.
<Preparation Example 6>
Sodium polyacrylate (degree of polymerization: 2,700-7,500, manufactured by Wako Pure Chemical Industries, Ltd.) was prepared. The said sodium polyacrylate was melt | dissolved in ion-exchange water so that it might become 5 mass%, and the high molecular weight body 6 solution was obtained.

<Comparative Preparation Example 1>
Polyethylene glycol 20000 (average molecular weight 20,000 ± 5,000, manufactured by Wako Pure Chemical Industries, Ltd.) was prepared. The polyethylene glycol 20000 was dissolved in ion-exchanged water so as to be 5% by mass to obtain a high molecular weight 7 solution.

  In the following Examples 1-1 to 1-7, enzymatic saccharification of holocelluloses in the biomass raw material of the present invention was performed using the high molecular weight solution obtained in Preparation Examples 1 to 6.

(Example 1-1)
Enzymatic saccharification was performed by preparing a mixed liquid by previously mixing the biomass raw material and the high molecular weight 1 solution, and adding a cellulose hydrolase to the mixed liquid to cause a reaction.

(I) Pulverization treatment of biomass raw material The cedar powder as a biomass raw material was pulverized using a tandem ring mill (trade name HV-30, TRU LLC). HV-30 has a crushing pot having an inner diameter of 284 mm and a material of S45C, and 10 S45C rings having an outer diameter of 252 mm, an inner diameter of 198 mm, and a thickness of 21 mm are stacked therein. Cedar powder pulverized to an average particle size of 200 μm using a dry pulverizer KDS-2 (manufactured by Kitagawa Iron Works Co., Ltd.) was charged into HV-30. Thereafter, the ring of HV-30 was moved at an amplitude of 8 mm and a frequency of 1,500 cpm (circle per minute) to obtain a cedar powder pulverized product having an average particle diameter of 20 μm. The particle size was measured using Microtrac MT3300EX2 (Nikkiso Co., Ltd.), which is a dedicated device for particle size measurement.

(Ii) Preparation of mixed liquid of biomass raw material and high molecular weight body (step (1))
As a biomass raw material, the cedar powder pulverized product obtained in (i) above, the polymer 1 solution and ion-exchanged water were mixed. The resulting mixture was autoclaved at 121 ° C. for 15 minutes. The liquid thus prepared was referred to as “cedar powder-high molecular weight mixture liquid”. In addition, it adjusted so that the density | concentration of the cedar powder ground material might be 20 mass%, and the density | concentration of the high molecular weight body 1 might be 0.5 mass% with the final density | concentration of the saccharification reaction liquid mentioned later.

(Iii) Enzymatic saccharification of biomass raw material (process (2))
CelliCTec2 (trade name) (manufactured by Novozyme) as a cellulose hydrolase was added to the cedar powder-high molecular weight mixture prepared in the above step (ii), and the protein amount per mass of the cedar powder pulverized product was 0.27. It added so that it might become mass%, and it was set as the saccharification reaction liquid. Enzymatic saccharification was performed by reacting the saccharification reaction solution at 50 ° C. for 48 hours while shaking at 120 rpm to obtain an enzyme saccharification solution.

  About the enzyme saccharified liquid obtained by (i)-(iii), the saccharification rate and the enzyme recovery rate were calculated | required by the method shown below. The results of saccharification rate and enzyme recovery rate are shown in Table 1 described later.

<Saccharification rate>
The saccharification rate was calculated by the following procedure by measuring the glucose concentration in the biomass raw material in step (1) and the glucose concentration in the enzyme saccharified solution.

(A) Glucose unit amount in 1 g of biomass raw material 0.6 mL of 72 mass% sulfuric acid was added to 1.0 g of biomass raw material (cedar powder pulverized product) and held at 30 ° C. for 1 hour, and then 16.8 mL of water was added. And then processed in an autoclave at 120 ° C. for 1 hour. About the sample after a process, the liquid quantity was measured (biomass raw material test liquid quantity). Thereafter, a glucose concentration was measured by an enzyme method (F-kit glucose: JK International) using a part of the sample. The glucose unit amount in 1 g of biomass raw material was calculated from the glucose concentration obtained in this measurement and the amount of biomass raw material test solution.
Glucose unit amount in 1 g of biomass raw material (g) = glucose concentration (mass / volume%) × biomass raw material test solution amount (mL) × 0.01

(B) Amount of glucose in enzyme saccharified solution For the enzyme saccharified solution, the precipitate and the supernatant were separated by centrifugation, and the amount of the recovered supernatant was measured (amount of saccharification test solution). The glucose concentration of the supernatant was measured by an enzyme method (F-kit glucose: JK International). The amount of glucose in the enzyme saccharified solution was calculated from the glucose concentration obtained in this measurement and the amount of saccharified test solution.
Glucose amount of enzyme saccharified solution (g) = glucose concentration (mass / volume%) × saccharified test solution amount (mL) × 0.01

(C) Measurement of saccharification rate From the amount of glucose obtained by (a) and (b) above and the amount of biomass raw material (cedar powder pulverized product) used in steps (1) and (ii), saccharification is performed according to the following formula: The rate was calculated. The results are shown in Table 1 described later.
Saccharification rate (%) = glucose amount of enzyme saccharified solution (g) / {glucose unit amount in 1 g of biomass raw material (g / g) × biomass raw material amount (g)} × 100

<Enzyme recovery rate 1>
The enzyme recovery rate 1 was calculated by the following procedure.
A sample after the enzyme saccharification treatment of (iii) above was prepared, and the precipitate and the supernatant liquid 1 were separated by centrifugation. The amount of protein in the obtained supernatant 1 was measured using a protein assay (trade name) (manufactured by Bio-Rad Laboratories). A blank solution 1 was a solution prepared according to Example 1-1 (i) to (iii) except that cedar powder pulverized product and cellulose hydrolase were not added. The solution prepared according to Example 1-1 (i) to (iii) was designated as enzyme solution 1 except that the cedar powder was not added. About the blank liquid 1 and the enzyme liquid 1, the protein amount was measured by the protein assay similarly to the above-mentioned.
The enzyme recovery rate was calculated from the following equation using the protein amounts of supernatant liquid 1, blank liquid 1, and enzyme liquid 1 obtained by measurement. The results are shown in Table 1 described later. The activity of the recovered cellulose hydrolase was confirmed by enzymatic saccharification using cedar powder pulverized material as a biomass raw material to produce sugar.
Enzyme recovery rate 1 (%) = (protein amount in supernatant solution 1−protein amount in blank solution 1) / (protein amount in enzyme solution 1−protein amount in blank solution 1) × 100

(Example 1-2)
Enzymatic saccharification was performed in the same manner as in Example 1-1 except that the high molecular weight 2 solution of Preparation Example 2 was used instead of the high molecular weight 1 solution of Preparation Example 1, and the saccharification rate and enzyme recovery rate were determined. The results are shown in Table 1 described later.

(Example 1-3)
Enzymatic saccharification was performed in the same manner as in Example 1-1 except that the high molecular weight 3 solution of Preparation Example 3 was used instead of the high molecular weight 1 solution of Preparation Example 1, and the saccharification rate and enzyme recovery rate were determined. The results are shown in Table 1 described later.

(Example 1-4)
Enzymatic saccharification was carried out in the same manner as in Example 1-1 except that the high molecular weight 4 solution of Preparation Example 4 was used instead of the high molecular weight 1 solution of Preparation Example 1, and the saccharification rate and enzyme recovery rate were determined. The results are shown in Table 1 described later.

(Example 1-5)
Enzymatic saccharification was carried out in the same manner as in Example 1-1 except that the high molecular weight product 5 solution of Preparation Example 5 was used instead of the high molecular weight product 1 solution of Preparation Example 1, and the saccharification rate and enzyme recovery rate were determined. The results are shown in Table 1 described later.

(Example 1-6)
Enzymatic saccharification was performed in the same manner as in Example 1-1 except that the high molecular weight 6 solution of Preparation Example 6 was used instead of the high molecular weight 1 solution of Preparation Example 1, and the saccharification rate and enzyme recovery rate were determined. The results are shown in Table 1 described later.

(Comparative Example 1-1)
Enzymatic saccharification was carried out in the same manner as in Example 1-1 except that the high molecular weight 1 solution of Preparation Example 1 was not added, and the saccharification rate and enzyme recovery rate were determined. The results are shown in Table 1 described later.

(Comparative Example 1-2)
Enzymatic saccharification was performed in the same manner as in Example 1-1 except that the high molecular weight 7 solution of Comparative Preparation Example 1 was used instead of the high molecular weight 1 solution of Preparation Example 1, and the saccharification rate and enzyme recovery rate were determined. . The results are shown in Table 1 described later.

(Example 1-7)
Cellulose hydrolase and high molecular weight 6 solution were mixed in advance to prepare a mixed solution, and biomass saccharification was performed by adding a biomass raw material to the mixed solution and causing the mixture to react. The specific method is shown below.

(I) Grinding treatment of biomass raw material A ground material of cedar powder was obtained in the same manner as in (1-1) of Example 1-1.

(Ii) Preparation of liquid mixture of cellulose hydrolase and high molecular weight (step (1))
Cellulose hydrolase CelliCec2 (trade name) (manufactured by Novozyme) was diluted with a high molecular weight 6 solution to prepare an “enzyme-high molecular weight liquid”. The final concentration of the saccharification reaction solution described later was adjusted so that the enzyme concentration was 0.27% by mass as the amount of protein per mass of cedar powder and the concentration of the high molecular weight body 6 was 0.5% by mass.

(Iii) Preparation of biomass raw material liquid A cedar powder pulverized product and ion-exchanged water were mixed. The solution was then autoclaved at 121 ° C. for 15 minutes. The liquid thus prepared was used as a biomass raw material liquid. The cedar powder pulverized product was adjusted to 20% by mass in the final concentration of the saccharification reaction solution described later.

(Iv) Enzymatic saccharification of biomass raw material (process (2))
The enzyme-high molecular weight body fluid obtained in (ii) was added to the biomass raw material fluid prepared in (iii) above to prepare a saccharification reaction solution. Enzymatic saccharification was performed by reacting the saccharification reaction solution at 50 ° C. for 48 hours while shaking at 120 rpm to obtain an enzyme saccharification solution.

  About the enzyme saccharification liquid obtained by process (i)-(iv), the saccharification rate and the enzyme collection | recovery rate were computed similarly to Example 1-1. The obtained results are shown in Table 1 described later.

(Comparative Example 1-3)
Enzymatic saccharification was performed in the same manner as in Example 1-7 except that skim milk was used at a final concentration of 2% instead of the high molecular weight 6 solution of Preparation Example 6, and the saccharification rate and enzyme recovery rate were determined. The results are shown in Table 1 described later.

The saccharification rates and enzyme recovery rates of Examples 1-1 to 1-7 and Comparative Examples 1-1 to 1-3 are shown in Table 1 below.

  In Example 2-1 below, ethanol was produced using the saccharide obtained by the saccharification method of the present invention.

(Example 2-1)
Cellulose hydrolase and high molecular weight 6 solution were mixed in advance to prepare a mixed solution, a biomass raw material was added to the mixed solution, and yeast was added to perform saccharification and alcohol fermentation simultaneously. The specific method is shown below.

(I) Grinding treatment of biomass raw material A ground material of cedar powder was obtained in the same manner as in (1-1) of Example 1-1.

(Ii) Preparation of liquid mixture of cellulose hydrolase and high molecular weight (step (1))
Cellulose hydrolase CelliCec2 (trade name) (manufactured by Novozyme) was diluted with a high molecular weight 6 solution to prepare an “enzyme-high molecular weight liquid”. The final concentration of the saccharification reaction solution described later was adjusted so that the enzyme concentration was 0.27% by mass as the amount of protein per mass of cedar powder and the concentration of the high molecular weight body 6 was 0.5% by mass.

(Iii) Preparation of biomass raw material liquid A cedar powder pulverized product and ion-exchanged water were mixed. The solution was then autoclaved at 121 ° C. for 15 minutes. The liquid thus prepared was used as a biomass raw material liquid. The cedar powder pulverized product was adjusted to 20% by mass in the final concentration of the saccharification reaction solution described later.

(Iv) Enzymatic saccharification and alcoholic fermentation of biomass raw material (process (2))
The enzyme-high molecular weight body fluid obtained in (ii) was added to the biomass raw material fluid prepared in (iii) above to prepare a saccharification reaction solution. Yeast (Shizosaccharomyces japonicus) 1.0 × 10 ⁹ cells / mL was added to 100 g of this saccharification reaction solution, and reacted at 37 ° C. for 72 hours with shaking at 80 rpm in a 500 mL baffled flask, and alcohol fermentation was performed simultaneously with enzymatic saccharification. .

About the sample after alcoholic fermentation obtained by process (i)-(iv), ethanol concentration and enzyme recovery were measured with the following method.
<Ethanol concentration>
The ethanol concentration was measured by the following procedure using an alcohol concentration meter. After completion of alcohol fermentation, the precipitate and the supernatant were separated by centrifugation. The ethanol concentration in the obtained supernatant was measured with Alcomate AL-3 (product name) (Woodson).
As a result, the ethanol concentration of the sample after alcohol fermentation was 61 mass / volume%.

<Enzyme recovery rate>
The enzyme recovery rate for the sample after alcohol fermentation was calculated according to the following procedure.
A sample after alcohol fermentation was prepared, and the precipitate 2 and the supernatant 2 were separated by centrifugation. The protein amount of the obtained supernatant 2 was measured using a protein assay (trade name) (manufactured by Bio-Rad Laboratories). A blank solution 2 was prepared in the same manner except that the biomass material and the enzyme were not added and the solution obtained by centrifugation. A solution prepared in the same manner except that the biomass raw material was not added and obtained by centrifugation was designated as enzyme solution 2. For the supernatant solution 2, the blank solution 2, and the enzyme solution 2, the protein amount was measured by a protein assay in the same manner as described above. The enzyme recovery rate 2 was calculated from the following formula.
Enzyme recovery rate 2 (%) = (protein amount in supernatant solution 2−protein amount in blank solution 2) / (protein amount in enzyme solution 2−protein amount in blank solution 2) × 100
The enzyme recovery rate for the sample after alcohol fermentation was 40%.

(Comparative Example 2-1)
Alcohol fermentation was performed according to Example 2-1, except that 2% of skim milk was used at the final concentration instead of the high molecular weight 6 solution for the preparation of the “enzyme-high molecular weight body fluid”. After the alcohol fermentation, ethanol concentration and enzyme recovery were measured.
As a result, the ethanol concentration after alcohol fermentation was 58% and the enzyme recovery rate was 4%.

(General)
As is clear from the results of Examples 1-1 to 1-7, according to the saccharification method of the present invention, poly (meth) acrylic acid (salt) is added to at least one of biomass raw material and cellulose hydrolase. By adding, adsorption | suction of the cellulose hydrolase by lignin can be suppressed, and the functional fall of the cellulose decomposing enzyme by this can be prevented. Therefore, by the saccharification method of the present invention, saccharides can be efficiently produced from holocelluloses contained in biomass raw materials.
In Example 1-7, the saccharification method of the present invention is also excellent in the recovery rate of cellulose hydrolase, enables reuse of cellulose hydrolase, and can produce saccharides efficiently. Show. Furthermore, as shown in Example 2-1, the saccharide obtained by the saccharification method of the present invention can be used effectively for the production of ethanol, and can also be used effectively for the production of other saccharides.
On the other hand, from the result of Comparative Example 1-1, when poly (meth) acrylic acid (salt) is not added, cellulose hydrolase is adsorbed on lignins, and the function of the cellulolytic enzyme is inhibited, and almost no cellulose hydrolysis is performed. It was found that the degradation enzyme cannot be recovered. Moreover, from the result of Comparative Example 1-2, when polyethylene glycol was used instead of poly (meth) acrylic acid (salt), cellulose hydrolase could hardly be recovered. This is probably because polyethylene glycol does not have an anionic functional group, so it did not interact with lignins or cellulose hydrolase, and adsorption of cellulose hydrolase by lignins could not be suppressed. From the result of Comparative Example 1-3, when skim milk is added, although the adsorption of cellulose hydrolase to lignin is slightly suppressed, the adsorption inhibiting action is not sufficient, and the recovery rate of cellulose hydrolase is not sufficient. There wasn't.

  The saccharification method of holocelluloses in biomass raw materials in which the lignins of the present invention coexist is one in which adsorption of cellulose hydrolase to lignins is remarkably suppressed, and saccharification of holocelluloses by cellulose hydrolase is suppressed. Without hindering, holocelluloses in the biomass material can be efficiently saccharified, which is useful. In addition, the cellulose hydrolase after the enzymatic saccharification reaction can be reused, and the cellulose hydrolase can be used efficiently, which is economically advantageous. In addition, the saccharide obtained by the saccharification method of the present invention can be used for alcohol fermentation, lactic acid fermentation, and the like, and can produce alcohol and ethanol using biomass raw material with high efficiency.

Claims (3)

A method for saccharification of holocelluloses in a biomass raw material in which lignins coexist, including the following steps (1) and (2):
Step (1): A step of adding poly (meth) acrylic acid (salt) in advance to at least one of the pulverized biomass raw material and cellulose hydrolase to obtain a mixed solution,
Step (2): By mixing the liquid mixture obtained in step (1) with cellulose hydrolase or pulverized biomass material, the biomass material and cellulose hydrolase are reacted, and the holocelluloses in the biomass material are reacted. Enzymatic saccharification process.
A method for producing ethanol from biomass raw materials in which lignins coexist, including the following steps (1) to (3):
Step (1): A step of adding poly (meth) acrylic acid (salt) in advance to at least one of the pulverized biomass raw material and cellulose hydrolase to obtain a mixed solution,
Step (2): By mixing the liquid mixture obtained in step (1) with cellulose hydrolase or pulverized biomass material, the biomass material and cellulose hydrolase are reacted, and the holocelluloses in the biomass material are reacted. Enzymatic saccharification, and
Step (3): A step of subjecting the saccharide to alcohol fermentation after the enzyme saccharification step of Step (2) or simultaneously with the enzyme saccharification step.
Furthermore, the manufacturing method of ethanol of Claim 2 including the process of collect | recovering the said cellulose hydrolase.