Saccharification enzyme composition, saccharification reaction liquid, and method for producing sugar

Abstract

A saccharification reaction solution in which at least one of cellulose and hemicellulose is saccharified, wherein the saccharifying enzyme and colloidal silica are contained in a dispersed state, and the proportion of the saccharifying enzyme not immobilized in the colloidal silica to the total saccharifying enzyme is 25% to 100%.

Description

Technical Field

[0001] This invention relates to a saccharifying enzyme composition, a saccharification reaction solution, and a method for manufacturing sugar. Background Technology

[0002] Cellulose-based bioethanol, which is produced from cellulose-based biomass containing cellulose or hemicellulose, has long been known.

[0003] As a method for producing sugars such as glucose from cellulosic biomass containing cellulose or hemicellulose (saccharification technology), the method of adding sulfuric acid to the cellulosic biomass for hydrolysis is known, but this has problems such as reactor corrosion and waste liquid treatment. In addition, for example, methods have been proposed to saccharify cellulosic biomass using solid acid catalysts with sulfonyl groups supported on carbon, zeolites, etc., but because it is a reaction between solids, the reaction rate is extremely slow, and there are also problems such as difficulty in separating unreacted residues from the solid acid catalyst. Furthermore, controlling hydrolysis in any of the above methods is difficult, and excessive reaction can lead to the decomposition of the sugar itself and a decrease in sugar yield.

[0004] On the other hand, a method for saccharification using enzymes is also known (see Patent Document 1). This method includes a hot water treatment step of treating the raw material with pressurized hot water, a mechanical pulverization step of mechanically pulverizing the hot water-treated material, and a saccharification step of saccharifying the mechanically pulverized material with enzymes. However, this method has problems such as a slow reaction rate when using enzymes for saccharification and insufficient concentration of the resulting saccharified solution.

[0005] Therefore, a method was proposed that allows the enzyme to exist in the reaction system at a higher concentration than in its dissolved state by supporting the enzyme on a silica-based mesoporous material, thereby enabling more efficient enzyme reactions (see Patent Document 2). However, this method has the problem of requiring a step of immobilizing the enzyme on the support, and the immobilized enzyme may have a reaction efficiency that is about 40-50% lower than that of the unimmobilized enzyme. Furthermore, because it involves a reaction between solids, there is also the problem of difficulty in separating the unreacted residue from the enzyme-immobilized support.

[0006] In addition, immobilized enzymes are known to be obtained by mixing silica sol with enzymes, forming silica gel, and then pulverizing the powder (see Patent Documents 3 and 4). While such immobilized enzymes can be recovered, the reaction efficiency itself is low. Furthermore, methods for hydrolyzing cellulose-containing plant fibers by mixing 0.5 μm to 100 μm silica powder with enzymes are also known, but these methods suffer from problems such as unclear effects of mixing silica powder and difficulty in separating unreacted residues from suspended silica powder (see Patent Document 5).

[0007] Existing technical documents

[0008] Patent documents

[0009] Patent Document 1: Japanese Patent Application Publication No. 2006-136263

[0010] Patent Document 2: Japanese Patent Application Publication No. 2009-125006

[0011] Patent Document 3: Japanese Patent Publication No. 63-2595

[0012] Patent Document 4: Japanese Patent Publication No. 63-21475

[0013] Patent Document 5: Japanese Patent Application Publication No. 10-66594 Summary of the Invention

[0014] The problem that the invention aims to solve

[0015] The present invention was made in view of the above facts, and the object is to provide a saccharifying enzyme composition, a saccharifying reaction solution, and a method for manufacturing sugar that improves the speed of saccharification reactions utilizing enzymes through a simple process.

[0016] Methods for solving problems

[0017] A first aspect of the invention for achieving the stated objective is a saccharification reaction solution, characterized in that it is a saccharification reaction solution in which at least one of cellulose and hemicellulose is saccharified, wherein the saccharifying enzyme and colloidal silica are contained in a dispersed state, and the proportion of the saccharifying enzyme not immobilized in the colloidal silica to the total saccharifying enzyme is 25% to 100%.

[0018] Preferably, the average primary particle size of the colloidal silica is 1 nm to 400 nm, and the particle size measured by dynamic light scattering method is greater than or equal to 5 nm and less than 500 nm.

[0019] Furthermore, the concentration of the saccharifying enzyme is preferably 0.005% to 3.0% by mass.

[0020] Furthermore, the concentration of the colloidal silica is preferably 0.005% to 40% by mass.

[0021] In addition, the preferred mass ratio of the saccharifying enzyme to the colloidal silica (saccharifying enzyme / colloidal silica) is 0.002 to 300.

[0022] In addition, a pH value of 3 to 11 is preferred.

[0023] In addition, the saccharifying enzyme preferably comprises at least one of a saccharifying enzyme derived from the genus Aspergillus and a saccharifying enzyme derived from the genus Trichoderma.

[0024] Furthermore, the second aspect of the present invention is a saccharifying enzyme composition, characterized in that it is a saccharifying composition of at least one of cellulose and hemicellulose, wherein the saccharifying enzyme is contained in a dispersed state, and colloidal silica with an average primary particle size of 1 nm to 400 nm and a particle size greater than or equal to 5 nm and less than 500 nm as determined by dynamic light scattering, and the proportion of the saccharifying enzyme not immobilized in the colloidal silica to the total saccharifying enzyme is 25% to 100%.

[0025] In addition, the third aspect of the present invention is a method for manufacturing sugar, characterized in that sugar is manufactured using the saccharification reaction solution described in any one of claims 1 to 7.

[0026] In addition, the preferred reaction temperature for the saccharification reaction is 5℃~100℃. Attached Figure Description

[0027] Figure 1 This study shows the pH dependence of the proportion of saccharifying enzymes not immobilized in colloidal silica relative to the total number of saccharifying enzymes in the saccharification composition.

[0028] Figure 2 This shows the pH dependence of the saccharification rate of the saccharification reaction solution after 14 days of enzymatic reaction.

[0029] Figure 3 This study shows the dependence of the proportion of unimmobilized glucoamylases on colloidal silica relative to the total glucoamylases in the glucoaming composition after 7 days of enzymatic reaction of the glucoaming reaction solution.

[0030] Figure 4 This shows the silica concentration dependence of the saccharification rate of the saccharification reaction solution after 14 days of enzymatic reaction.

[0031] Figure 5 This study demonstrates the dependence of the saccharification rate of the saccharification reaction solution after 14 days of enzymatic reaction on the saccharification rate within the range of 0.1–300 for the saccharifying enzyme / colloidal silica ratio.

[0032] Figure 6 This study showed the dependence of the saccharification rate of the saccharification reaction solution after 14 days of enzymatic reaction on the saccharification rate within the range of 0.003 to 0.1 for the saccharifying enzyme / colloidal silica ratio.

[0033] Figure 7 This shows the mean primary particle size dependence of the saccharification rate of the saccharification reaction solution after 14 days of enzymatic reaction. Detailed Implementation

[0034] In this invention, at least one of cellulose and hemicellulose is used as a raw material.

[0035] This cellulose or hemicellulose is found in cellulosic biomass such as broadleaf trees, coniferous trees, and other agricultural, forestry, and aquatic resources, or waste products from such resources. More specifically, examples include sugarcane bagasse, rice straw, corn stalks, empty fruit bunches of oil palm, wood fiber, wood chips, veneer shavings, wood flour, pulp, waste paper, cotton, sea squirts, and acetic acid bacteria. Furthermore, there are no particular limitations on these raw materials as long as they are derived from cellulosic biomass; one type can be used alone, or two or more can be used in combination.

[0036] The preferred ingredients are cellulose or hemicellulose contained in eucalyptus wood powder (broadleaf tree), cedar wood powder (coniferous tree), sugarcane bagasse, rice straw, corn stalks, empty fruit bunches of oil palm, and cotton. Although the reason is uncertain, these ingredients facilitate decellulose removal and allow for the extraction of sugar at a relatively high yield.

[0037] Cellulose refers to a polymer formed by the polymerization of glucose through β-1,4 glycosidic bonds. Hemicellulose refers to a polymer formed by the polymerization of glucose, xylose, mannose, galactose, etc., through glycosidic bonds; it refers to water-insoluble polysaccharides other than cellulose.

[0038] In addition, cellulose may also contain cellulosic sugars and cellobioses as partial decomposition products, and these can be crystalline or amorphous. Furthermore, they can be carboxymethylated, aldehyde-modified, or esterified derivatives. Moreover, as mentioned above, cellulose or hemicellulose is not particularly limited as long as it is derived from biomass; it can originate from plants, fungi, or bacteria.

[0039] As the saccharifying enzyme of the present invention, a saccharifying enzyme mainly composed of cellulase is used. This cellulase refers to an enzyme that breaks down cellulose or hemicellulose into sugars such as glucose.

[0040] The microorganisms that produce this saccharifying enzyme are not particularly limited, but can be listed as, for example, *Acremonium*, *Aspergillus*, *Chaetomium*, *Fusarium*, *Humicola*, *Irpex*, *Phanerochaete*, *Penicillium*, *Schizophyllum*, *Sporotrichum*, *Trametes*, and *Trichoderma*. In addition to *Trichoderma*, other examples include bacteria such as *Clostridium*, *Pseudomonas*, *Cellulomonas*, *Ruminococcus*, *Bacillus*, *Sulfolobus*, *Streptomyces*, *Thermoactinomyces*, and *Thermomonospora*. Furthermore, these enzymes can be artificially modified. They can be used alone or in combination.

[0041] Enzymes derived from the genera *Aspergillus* and *Trichoderma* are particularly preferred due to their high activity on crystalline cellulose.

[0042] In addition, cellulase can be a group of enzymes. Examples of such enzymes include endoglucanase (EC 3.2.1.74), cellobiase (EC 3.2.1.91), and β-glucosidase (EC 23.2.4.1, EC 3.2.1.21). Furthermore, it is preferable to use cellulases derived from different microorganisms in combination. In this case, their synergistic effect can further promote the saccharification of cellulose or hemicellulose.

[0043] Most of the cellulases mentioned above have optimal enzyme activity in the pH range of 3 to 6, but some, known as alkaline cellulases, have optimal enzyme activity in the pH range of 6 to 10. Additionally, most of the cellulases mentioned above have optimal enzyme activity in the reaction temperature range of 25°C to 50°C, but some, known as thermostable cellulases, have optimal enzyme activity in the temperature range of 70°C to 100°C.

[0044] In this invention, colloidal silica has an average primary particle size of 1 nm to 400 nm, preferably 5 nm to 350 nm, and is dispersed in the saccharification reaction solution. The average primary particle size is determined by the specific surface area S (m²) measured by the nitrogen adsorption method (BET method). 2 The particle size ( / g) is calculated using the conversion formula D(nm) = 2720 / S. The colloidal silica, partially or entirely, serves as a carrier for the saccharifying enzyme and is used in a dispersed state together with the saccharifying enzyme not immobilized in the colloidal silica. The particle size of the colloidal silica in the dispersed liquid is expressed by the particle size measured using dynamic light scattering. In this invention, it is desirable that the particle size measured by dynamic light scattering is greater than or equal to 5 nm and less than 500 nm, preferably 10 nm to 450 nm. Furthermore, the silica is not porous but solid. In addition, the colloidal silica can be prepared into a dispersion in a dispersion medium such as water, methanol, ethanol, acetone, methyl ethyl ketone, or ethylene glycol; the dispersion is referred to as a colloidal solution or sol. The dispersion medium can be selected within a range that does not inhibit enzyme activity, but water and ethanol are preferred.

[0045] On the other hand, silica powder known as sedimentation silica is a porous powder with an average primary particle size of less than 400 nm and a particle size of more than 500 nm as determined by dynamic light scattering. Even when suspended in a dispersion medium, it does not exhibit the properties of a colloidal solution and does not have the high dispersibility of the colloidal silica of the present invention.

[0046] Methods for manufacturing colloidal silica include the water glass method using water glass as a raw material, the alkoxide method using metal alkoxides as raw materials, and the gas-phase method using silicon chloride compounds as raw materials. Colloidal silica obtained by any of these methods can be used, but the water glass method is preferred.

[0047] The saccharification reaction solution of the present invention is prepared by dispersing saccharifying enzymes and colloidal silica using at least one of cellulose and hemicellulose as raw materials, wherein the proportion of saccharifying enzymes not immobilized in colloidal silica relative to the total saccharifying enzymes is 25% to 100%. More preferably, the proportion of saccharifying enzymes not immobilized in colloidal silica relative to the total saccharifying enzymes is 50% to 100%. If the proportion of saccharifying enzymes not immobilized in colloidal silica relative to the total saccharifying enzymes is less than 25%, the reaction efficiency deteriorates, and therefore it is not preferred.

[0048] In the saccharification reaction solution, the concentration of the saccharifying enzyme is 0.005% to 3.0% by mass, preferably 0.01% to 1.0% by mass. If the concentration of the saccharifying enzyme is less than 0.005% by mass, the reaction efficiency decreases and it is not preferred. On the other hand, if it is greater than 3.0% by mass, not only is the saccharifying enzyme difficult to dissolve in the solution, but it is also not economically suitable.

[0049] Furthermore, in the saccharification reaction solution, the concentration of colloidal silica is 0.005% to 40% by mass, preferably 0.01% to 10% by mass. If the concentration of colloidal silica is less than 0.005% by mass, the reaction efficiency decreases and it is not preferred; on the other hand, if it is greater than 40% by mass, not only does the dispersibility deteriorate, but it is also economically unsuitable.

[0050] Furthermore, in the saccharification reaction solution, the mass ratio of saccharifying enzyme to colloidal silica (saccharifying enzyme / colloidal silica) is 0.002 to 300, preferably 0.1 to 10 or less. If the mass ratio deviates from this range, the improvement in reaction efficiency becomes insignificant.

[0051] Furthermore, the pH value of the saccharification reaction solution is 3 to 11, preferably 3 to 9, and more preferably 4 to 7. If the pH value is below 3, the colloidal silica will aggregate, reducing the reaction efficiency of the saccharifying enzyme. On the other hand, if the pH value is above 11, the colloidal silica becomes easily soluble, which is not preferred.

[0052] Examples of pH adjusters for the saccharification reaction solution include inorganic acids such as sulfuric acid, hydrochloric acid, and nitric acid; carboxylic acids such as acetic acid and oxalic acid; hydroxy acids such as citric acid, tartaric acid, and malic acid; hydroxide bases such as sodium hydroxide and potassium hydroxide; ammonia; and urea. The type and concentration of these pH adjusters are not particularly limited, as long as they do not impair the effects of the present invention. Furthermore, these pH adjusters can be used alone or in combination of two or more. They can also be used as a buffer solution with a buffering effect.

[0053] Furthermore, the reaction temperature of the saccharification reaction solution of the present invention is 5°C to 100°C, preferably 20°C to 55°C. If the reaction temperature is below 5°C, the efficiency of the saccharification reaction will be significantly reduced; if the temperature is above 100°C, the saccharifying enzyme may be inactivated, which is not preferred.

[0054] Furthermore, the pretreatment of cellulosic biomass containing cellulose or hemicellulose can be carried out within a known scope. Generally, it can be used as a raw material for saccharification reactions by physically crushing it using a mill or similar means, or by chemically destroying the structure of lignin, cellulose, and hemicellulose using acid or alkali treatment.

[0055] When preparing the saccharification reaction solution, colloidal silica can be added to the reaction solution in which the saccharifying enzyme is dispersed, or the saccharifying enzyme can be added to the reaction solution in which colloidal silica is dispersed. Other additives, such as pH adjusters, can be added in any order, as long as they do not inhibit the effects of this invention.

[0056] During the saccharification reaction, reverse osmosis (RO) membranes or ultrafiltration (UF) membranes with appropriate molecular weight cutoffs can be used to separate the saccharifying enzymes and colloidal silica from sugars such as glucose. A molecular weight cutoff of 1000 to 100000 is preferred. If the molecular weight cutoff is less than this range, although sugar separation is possible, clogging is likely, and the membrane permeation rate will be significantly reduced. Conversely, if the molecular weight cutoff is greater than this range, the saccharifying enzymes and colloidal silica may flow out along with the sugars, which is not preferable.

[0057] Example

[0058] The following description is based on embodiments, but the invention is not limited to these embodiments in any way.

[0059] The average primary particle size of colloidal silica and the particle size determined by dynamic light scattering were measured using the following measuring apparatus.

[0060] Nitrogen adsorption method measurement device (measurement of average primary particle size): Monosorb MS-16 (manufactured by Korniron Contract Co., Ltd.)

[0061] Dynamic light scattering particle size distribution apparatus: Zetasizer Nano S (made by Malubarn Instrument Mentz)

[0062] (Examples 1-7, Comparative Example 1: Glycoamylase composition)

[0063] First, prepare an aqueous solution of cellulase as a saccharifying enzyme aqueous solution according to the following steps. Furthermore, the cellulase used is a cellulase derived from the genus *Aspergillus niger* (prepared by MP biomedicals), which has optimal enzymatic activity in the pH range of 3–6.

[0064] First, 15g of cellulase powder was added to 85g of deionized water and dissolved at room temperature using a magnetic stirrer for 2 hours while stirring, to obtain a 15% by mass cellulase aqueous solution. Next, 200g of alkaline silica sol (pH 9.0, silica concentration 40% by mass) obtained by dispersing solid spherical colloidal silica (average primary particle size: 35nm, particle size determined by dynamic light scattering: 55nm) in water was treated with a strong acidic hydrogen-form cation exchange resin, IR-120B (manufactured by Organo), to remove alkali metal ions, to obtain 200g of acidic silica sol (pH 2.1, silica concentration 40% by mass). While stirring, 4.0 g of the above-mentioned cellulase aqueous solution was added to 15 g of the obtained acidic silica sol. Then, to adjust the pH, 1.0 g of one of the following was added: 1 M equivalent of acetic acid, sodium acetate (hereinafter sometimes referred to as Na acetate), acetate-Na acetate buffer (pH 3.5–5.0), 0.5 M equivalent of NaOH, or HCl. This yielded a saccharifying enzyme composition with a silica concentration of 30% by mass and a cellulase concentration of 3% by mass. The particle size of the obtained saccharifying enzyme composition was confirmed using dynamic light scattering, and the result was 55 nm, indicating no change in particle size within the colloidal silica dispersion.

[0065] (The saccharifying enzyme composition of Example 8)

[0066] In a solution of 15g of alkaline silica sol (pH 9.0, silica concentration 40% by mass) obtained by dispersing solid spherical colloidal silica (average primary particle size: 35nm, particle size measured by dynamic light scattering: 55nm) prepared by the water glass method in water, 4.0g of the above-mentioned cellulase aqueous solution was added while stirring. Then, to adjust the pH, 0.5g of 1M equivalent Na acetate and 0.5g of 1M equivalent NaOH were added to obtain a saccharifying enzyme composition with a silica concentration of 30% by mass and a cellulase concentration of 3% by mass. The particle size of the obtained saccharifying enzyme composition was confirmed to be 55nm by dynamic light scattering, indicating no change in the particle size within the colloidal silica dispersion. Table 1 shows the saccharifying enzyme compositions of Examples 1-8.

[0067] (Determination of the ratio of saccharifying enzymes not immobilized in colloidal silica to all saccharifying enzymes)

[0068] In the saccharifying enzyme composition of the present invention, the ratio of saccharifying enzyme not immobilized on colloidal silica to the total saccharifying enzyme is calculated by quantitatively measuring the concentration of saccharifying enzyme in the supernatant obtained by centrifugation using the Bradford method (Coomassie Brilliant Blue method, CBB method).

[0069] The specific steps are as follows.

[0070] 1.0 mL of the saccharifying enzyme composition was taken into a 50 mL centrifuge sedimentation tube and centrifuged using a high-speed cooled SRX-201 centrifuge (manufactured by Tommy Seiko Co., Ltd.) at 25,000 G, 30 minutes, and 4 °C to obtain the supernatant. Next, 2.5 mL of a protein analysis CBB solution (5x concentrated) (manufactured by Nakalitesk) diluted 5 times with deionized water was added to a disposable sample cell with a cell length of 10 mm. Then, 0.05 mL of the supernatant was added, and the cell was sealed. The mixture was repeatedly inverted to ensure uniform mixing. After standing for 30 minutes, the absorbance was measured at a wavelength of 595 nm using a UV-3150 spectrophotometer (manufactured by Shimadzu Corporation). A standard curve was prepared by similarly measuring the absorbance of samples with known saccharifying enzyme concentrations. The saccharifying enzyme concentration in the supernatant was calculated from the obtained standard curve.

[0071] The concentration of saccharifying enzyme in the supernatant obtained by the above method is divided by the concentration of added saccharifying enzyme and then multiplied by 100. This value is taken as the ratio of saccharifying enzyme not immobilized on colloidal silica to the total saccharifying enzyme. The relationship between the ratio of saccharifying enzyme not immobilized on colloidal silica to the total saccharifying enzyme in the saccharifying enzyme compositions of Examples 1-8 and pH value is shown below. Figure 1 The ratio of unimmobilized saccharifying enzymes to total saccharifying enzymes depends on the pH of the saccharifying enzyme composition.

[0072] (Comparative Examples 2-10: Saccharifying enzyme compositions)

[0073] Except that colloidal silica was not added and deionized water was used instead to adjust the concentration of the saccharifying enzyme, the process was carried out in the same manner as in Examples 1-8 to obtain a saccharifying enzyme composition that does not contain colloidal silica. This saccharifying enzyme composition is shown in Table 1.

[0074] (Comparative Example 11: Glycoamylase composition)

[0075] 20g of an alkaline silica sol (pH 9.0, silica concentration 40% by mass) obtained by dispersing solid spherical colloidal silica (average primary particle size: 35nm, particle size measured by dynamic light scattering: 55nm) in water was treated with a strong acidic hydrogen-form cation exchange resin, IR-120B (manufactured by Organo), to remove alkali metal ions, yielding 20g of an acidic silica sol (pH 2.1, silica concentration 40% by mass). 5g of deionized water was added to 15g of the obtained acidic silica sol to obtain a saccharifying enzyme composition with a silica concentration of 30% by mass. The saccharifying enzyme composition is shown in Table 1.

[0076] (Saccharification reaction solutions of Examples 1-8)

[0077] 2.5% by mass of microcrystalline cellulose powder was added to the saccharifying enzyme compositions of Examples 1-8 and dispersed to prepare a saccharification reaction solution.

[0078] The specific steps are as follows.

[0079] Add 10 mL of each saccharifying enzyme composition to a 20 mL glass bottle, and then use... While stirring with a 10mm stir bar, add 0.25g of microcrystalline cellulose powder (manufactured by MP biomedicals) (equivalent to 25mg / mL), and then seal.

[0080] (Methods for manufacturing sugar in Examples 1-8)

[0081] The saccharification reaction solutions of Examples 1 to 8 were subjected to enzymatic reaction for 14 days in a constant temperature room at 24°C with stirring.

[0082] The reaction temperature of the enzymatic reaction in the saccharification reaction solution, and the results of the saccharification rate determination after 3 days and 14 days are shown in Table 2. The relationship between the saccharification rate and pH value after 14 days of enzymatic reaction in the saccharification reaction solutions of Examples 1-8 is shown in... Figure 2 .

[0083] (Comparative Examples 1-11)

[0084] The preparation of the saccharification reaction solution and the method of sugar production were carried out in the same manner as in Examples 1-8. The results are shown in Table 2. Furthermore, the relationship between the saccharification rate and pH value of the saccharification reaction solutions of Comparative Examples 1-8 after 14 days of enzymatic reaction is shown in Table 2. Figure 2 .Depend on Figure 1 , Figure 2 It was determined that, in each pH range, compared with individual saccharifying enzymes, saccharifying enzymes combined with colloidal silica but not immobilized in colloidal silica had a higher saccharification rate in saccharification reaction solutions with a ratio of 42% to 100% of all saccharifying enzymes.

[0085] (Determination of saccharification rate)

[0086] The concentration of glucose generated during the enzymatic reaction of the saccharification reaction solution was quantified using the enzymatic method (G6PDH-HK method), and the saccharification rate was calculated.

[0087] A 0.5 mL sample of the saccharification reaction solution was taken onto a 2 mL microchip and heated at 110 °C for 30 minutes to inactivate the enzyme. Next, to remove unreacted cellulose and colloidal silica, the sample was transferred to a 50 mL centrifuge sedimentation tube and centrifuged using a high-speed cooled SRX-201 centrifuge (manufactured by Tommy Seiko Co., Ltd.) at 25,000 G, 30 minutes, and 4 °C. The supernatant was immediately recovered after centrifugation. The enzymatic method was performed using an F-kit glucon (manufactured by JK Internason). The absorbance at 340 nm (cell length 10 mm) was measured using a UV-3150 spectrophotometer (manufactured by Shimadzu Corporation).

[0088] The specific steps are as follows.

[0089] Add 1.0 mL of F-kit solution I to a disposable sample cell with a cell length of 10 mm. Next, add 0.1 mL of the supernatant and 1.9 mL of deionized water, and seal the cell. Then, repeatedly invert the solution to mix thoroughly. After standing for 3 minutes, measure the absorbance of the supernatant at 340 nm using a spectrophotometer, which is recorded as E1. Next, add 0.02 mL of F-kit solution II, and repeatedly invert the solution to mix thoroughly. After standing for 15 minutes, measure the absorbance of the supernatant at 340 nm using a spectrophotometer, which is recorded as E2. The absorbance of the blank is the value obtained by measuring with deionized water instead of the supernatant.

[0090] The concentration of D-glucose is obtained by the following formula.

[0091]

[0092] ΔE = (E2 - E1)sample - (E2 - E1)blank

[0093] V (volume of reaction solution): 3.02 [mL]

[0094] Mw (molecular weight): 180.16 [g / mol]

[0095] d (optical path length): 1 [cm]

[0096] ε (absorption coefficient): 6.3 [mmol] -1 ·cm -1 ]

[0097] v (sample size): 0.1 [mL]

[0098] The saccharification rate is obtained by dividing the concentration of glucose produced during the enzyme reaction of the saccharification reaction solution determined by the above method by the concentration of added microcrystalline cellulose powder (equivalent to 25 mg / mL) and then multiplying by 100.

[0099] [Table 1]

[0100]

[0101] pH adjuster

[0102] A: Acetic acid

[0103] B: Sodium acetate buffer solution (pH = 3.5)

[0104] C: Sodium acetate buffer (pH = 4.0)

[0105] D: Sodium acetate buffer solution (pH = 4.5)

[0106] E: Sodium acetate buffer (pH = 5.0)

[0107] F: Sodium acetate

[0108] G: NaOH

[0109] H: HCl

[0110] [Table 2]

[0111]

[0112] (Saccharifying enzyme compositions of Examples 9-26)

[0113] First, an aqueous solution of cellulase and an aqueous solution of saccharifying enzyme are prepared through the following steps. Furthermore, the cellulase used is a cellulase derived from the genus *Aspergillus niger* (prepared by MP biomedicals), which exhibits optimal enzymatic activity in the pH range of 3–6.

[0114] First, 10g of cellulase powder was added to 90g of deionized water and dissolved at room temperature using a magnetic stirrer for 2 hours while stirring, to obtain a 10% (w / w) aqueous solution of cellulase. Next, 200g of alkaline silica sol (pH 9.0, silica concentration 40% (w / w)) obtained by dispersing solid spherical colloidal silica (average primary particle size: 35nm, particle size determined by dynamic light scattering: 55nm) in water was treated with a strong acidic hydrogen-form cation exchange resin, IR-120B (manufactured by Organo), to remove alkali metal ions, to obtain 200g of acidic silica sol (pH 2.1, silica concentration 40% (w / w)). Deionized water and the aforementioned 10% (w / w) cellulase aqueous solution were added to the obtained acidic silica sol while stirring. Then, to adjust the pH, 0.05 M equivalents of 1 M acetate-sodium acetate buffer (pH 4.0) were added to obtain a saccharifying enzyme composition with a silica concentration of 0.01–38% (w / w) and a cellulase concentration of 0.1–3% (w / w). The particle size of the obtained saccharifying enzyme composition was confirmed using dynamic light scattering, and the result was 55 nm, indicating no change in particle size within the colloidal silica dispersion.

[0115] Table 3 shows the saccharifying enzyme compositions of Examples 9–26.

[0116] (Comparative Examples 12-14: glucoamylase compositions)

[0117] Except that colloidal silica was not added, and deionized water was used instead to adjust the concentration of the saccharifying enzyme, the procedure was the same as in Examples 9-26. The saccharifying enzyme compositions are shown in Table 3.

[0118] The preparation of the saccharification reaction solution, the method of sugar production, and the determination of saccharification reaction efficiency were carried out in the same manner as in Examples 1-8. The results of the saccharification rate determination are shown in Table 4. Furthermore, the relationship between the saccharification rate and silica concentration after 14 days of enzymatic reaction of the saccharification reaction solutions of Examples 3, 9-13, and Comparative Example 4 is shown in Table 4. Figure 4 In saccharification reaction solutions where the proportion of saccharifying enzymes not immobilized in colloidal silica to the total saccharifying enzymes was 25% to 100%, high saccharification rates were observed in the range of 0.01% to 30% by mass of silica concentration, which was also higher than the saccharification rate without colloidal silica. In Example 22, a silica concentration of 38% by mass also showed a high saccharification rate, which was also higher than the saccharification rate without colloidal silica.

[0119] It was further determined that in a saccharification reaction solution where the proportion of saccharifying enzymes not immobilized in colloidal silica to all saccharifying enzymes was 25% to 100%, a high saccharification rate was observed in the range of 0.1% to 3.0% by mass of saccharifying enzyme concentration, which was also higher than that in the case without colloidal silica.

[0120] In addition, the relationship between the saccharification rate and the mass ratio of saccharifying enzyme to colloidal silica (saccharifying enzyme / colloidal silica) of the saccharification reaction solutions of Examples 3, 9-13, and Comparative Example 4 after 14 days of enzymatic reaction is shown in the figure. Figure 5 .

[0121] The glycation rates of the glycation reaction solutions of Examples 22-26 and Comparative Example 14 after 14 days of enzymatic reaction, and the relationship between the mass ratio of glycans to colloidal silica (glycans / colloidal silica) are shown in the figures. Figure 6 .

[0122] Depend on Figure 5 , 6 It was determined that in a saccharification reaction solution where the proportion of saccharifying enzymes not immobilized in colloidal silica to the total saccharifying enzymes was 25% to 100%, a high saccharification rate was observed in the range of 0.003 to 300 mass ratio of saccharifying enzymes to colloidal silica (saccharifying enzyme / colloidal silica), which was also higher than that in the case without colloidal silica.

[0123] Furthermore, the relationship between the saccharification rate and primary particle size of the saccharification reaction solutions of Examples 20, 27-31, and Comparative Example 13 after 14 days of enzymatic reaction is shown in the figure. Figure 7 In saccharification reaction solutions where the proportion of saccharifying enzymes not immobilized in colloidal silica to all saccharifying enzymes is 25%–100%, the average primary particle size of the colloidal silica is 5 nm–310 nm, resulting in a high saccharification rate, which is also higher than that in the absence of colloidal silica.

[0124] [Table 3]

[0125]

[0126] [Table 4]

[0127]

[0128] (The saccharifying enzyme composition of Example 27)

[0129] First, an aqueous solution of cellulase is prepared as an aqueous solution of saccharifying enzyme through the following steps. Furthermore, the cellulase used is a cellulase derived from the genus *Aspergillus niger* (prepared by MP biomedicals), which exhibits optimal enzymatic activity in the pH range of 3–6.

[0130] Next, 10g of acidic silica sol (pH 2.8, silica concentration 10% by mass) obtained by dispersing solid spherical colloidal silica (average primary particle size: 5nm, particle size measured by dynamic light scattering: 15nm) manufactured by the water glass method in water was added with stirring. Then, 8.0g of deionized water and 1.0g of the aforementioned 10% by mass cellulase aqueous solution were added. To adjust the pH, 1.0g of 1M equivalent acetate-sodium acetate buffer (pH 4.0) was added, resulting in a saccharifying enzyme composition with a silica concentration of 5% by mass and a cellulase concentration of 0.5% by mass. The particle size of the obtained saccharifying enzyme composition was confirmed by dynamic light scattering to be 15nm, indicating no change in the particle size within the colloidal silica dispersion.

[0131] (The saccharifying enzyme composition of Example 28)

[0132] Acidic silica sol (pH 2.6, silica concentration 20% by mass) obtained by dispersing 5.0 g of solid spherical colloidal silica (average primary particle size: 12 nm, particle size determined by dynamic light scattering: 20 nm) prepared by the water glass method (pH 2.6, silica concentration 20% by mass) in water was added with stirring. 13.0 g of deionized water and 1.0 g of the aforementioned 10% by mass cellulase aqueous solution were then added. To adjust the pH, 1.0 g of 1 M equivalent acetate-sodium acetate buffer (pH 4.0) was added, resulting in a saccharifying enzyme composition with a silica concentration of 5% by mass and a cellulase concentration of 0.5% by mass. The particle size of the obtained saccharifying enzyme composition was confirmed by dynamic light scattering to be 20 nm, indicating no change in the particle size within the colloidal silica dispersion.

[0133] (The saccharifying enzyme composition of Example 29)

[0134] 20g of alkaline silica sol (pH 9.5, silica concentration 40% by mass), obtained by dispersing solid spherical colloidal silica (average primary particle size: 80nm, particle size measured by dynamic light scattering: 120nm) in water using the water glass method, was treated with a strong acidic hydrogen-form cation exchange resin, IR-120B (manufactured by Organo), to remove alkali metal ions, yielding 20g of acidic silica sol (pH 2.0, silica concentration 40% by mass). While stirring, 15.5g of deionized water and 1.0g of the aforementioned 10% by mass cellulase aqueous solution were added to 2.5g of the obtained acidic silica sol. Furthermore, to adjust the pH, 1.0g of 1M equivalent acetate-sodium acetate buffer (pH 4.0) was added, resulting in a saccharifying enzyme composition with a silica concentration of 5% by mass and a cellulase concentration of 0.5% by mass. The particle size of the obtained saccharifying enzyme composition was confirmed by dynamic light scattering, and the result was 120 nm. The particle size in the colloidal silica dispersion did not change.

[0135] (The saccharifying enzyme composition of Example 30)

[0136] 20g of alkaline silica sol (pH 9.3, silica concentration 40% by mass) obtained by dispersing solid spherical colloidal silica (average primary particle size: 160nm, particle size measured by dynamic light scattering method: 200nm) in water was treated with a strong acidic hydrogen-type cation exchange resin, IR-120B (manufactured by Organo), to remove alkali metal ions, thereby obtaining 20g of acidic silica sol (pH 2.3, silica (SiO2) concentration 40% by mass). While stirring, 15.5 g of deionized water and 1.0 g of the aforementioned 10% (w / w) cellulase aqueous solution were added to 2.5 g of the obtained acidic silica sol. Then, to adjust the pH, 1.0 g of 1 M equivalent acetate-sodium acetate buffer (pH 4.0) was added to obtain a saccharifying enzyme composition with a silica concentration of 5% (w / w) and a cellulase concentration of 0.5% (w / w). The particle size of the obtained saccharifying enzyme composition was confirmed using dynamic light scattering, and the result was 200 nm, indicating no change in particle size within the colloidal silica dispersion.

[0137] (The saccharifying enzyme composition of Example 31)

[0138] 20g of alkaline silica sol (pH 8.5, silica concentration 40% by mass), obtained by dispersing solid spherical colloidal silica (average primary particle size: 310nm, particle size measured by dynamic light scattering: 450nm) in water via the water glass method, was treated with a strong acidic hydrogen-form cation exchange resin, IR-120B (manufactured by Organo), to remove alkali metal ions, yielding 20g of acidic silica sol (pH 3.3, silica concentration 40% by mass). While stirring, 15.5g of deionized water and 1.0g of the aforementioned 10% by mass cellulase aqueous solution were added to 2.5g of the obtained acidic silica sol. Furthermore, to adjust the pH, 1.0g of 1M equivalent acetate-sodium acetate buffer (pH 4.0) was added, resulting in a saccharifying enzyme composition with a silica concentration of 5% by mass and a cellulase concentration of 0.5% by mass. The particle size of the obtained saccharifying enzyme composition was confirmed by dynamic light scattering, and the result was 450 nm. The particle size in the colloidal silica dispersion did not change.

[0139] Table 5 shows the saccharifying enzyme compositions of Examples 27–31.

[0140] The preparation of the saccharification reaction solution, the method of sugar production, and the determination of saccharification reaction efficiency were carried out in the same manner as in Examples 1-8. The results of the saccharification rate determination are shown in Table 6.

[0141] [Table 5]

[0142]

[0143] [Table 6]

[0144]

[0145] (Saccharifying enzyme compositions of Examples 32-37)

[0146] First, an aqueous solution of cellulase is prepared as an aqueous solution of saccharifying enzyme through the following steps. Furthermore, the cellulase used is a cellulase derived from the genus *Trichoderma reesei* (prepared by Sigma Aldrich) that exhibits optimal enzymatic activity in the pH range of 3–6.

[0147] First, 1g of cellulase powder was added to 9g of deionized water and dissolved at room temperature using a magnetic stirrer for 2 hours while stirring, to obtain a 10% (w / w) aqueous solution of cellulase. Next, 20g of alkaline silica sol (pH 9.0, silica concentration 40% (w / w)) obtained by dispersing solid spherical colloidal silica (average primary particle size: 35nm, particle size determined by dynamic light scattering: 55nm) in water was treated with a strong acidic hydrogen-form cation exchange resin, IR-120B (manufactured by Organo), to remove alkali metal ions, to obtain 20g of acidic silica sol (pH 2.1, silica concentration 40% (w / w)). Deionized water and the aforementioned 10% (w / w) cellulase aqueous solution were added to the obtained acidic silica sol while stirring. Then, to adjust the pH, 0.05 M equivalents of 1 M acetate-sodium acetate buffer (pH 4.0) were added to obtain a saccharifying enzyme composition with a silica concentration of 0.01–10% (w / w) and a cellulase concentration of 0.01–1% (w / w). The particle size of the obtained saccharifying enzyme composition was confirmed using dynamic light scattering, and the result was 55 nm, indicating no change in particle size within the colloidal silica dispersion.

[0148] Table 7 shows the saccharifying enzyme compositions of Examples 32-37.

[0149] (Comparative Examples 15-20: glucoamylase compositions)

[0150] Except that colloidal silica was not added, and deionized water was used instead to adjust the concentration of the saccharifying enzyme, the procedure was the same as in Examples 32-37. The saccharifying enzyme compositions are shown in Table 7.

[0151] The preparation of the saccharification reaction solution, the method of sugar production, and the determination of saccharification reaction efficiency were carried out in the same manner as in Examples 1-8. The results of the saccharification rate determination are shown in Table 8.

[0152] In saccharification reaction solutions where the proportion of saccharifying enzymes not immobilized in colloidal silica to all saccharifying enzymes is 25%–100%, cellulases derived from the genus *Trichoderma reesei* show a similarly high saccharification rate as cellulases derived from the genus *Aspergillus niger*, and also a higher saccharification rate than in the absence of colloidal silica.

[0153] Furthermore, it was determined that in saccharification reaction solutions where the proportion of saccharifying enzymes not immobilized in colloidal silica to all saccharifying enzymes was 25% to 100%, a high saccharification rate was observed in the range of silica concentration from 0.01% to 10% by mass, which was also higher than the saccharification rate in the absence of colloidal silica.

[0154] It was further determined that in a saccharification reaction solution where the proportion of saccharifying enzymes not immobilized in colloidal silica to all saccharifying enzymes was 25% to 100%, a high saccharification rate was observed in the range of 0.01 to 1.0% by mass of saccharifying enzyme concentration, which was also higher than that in the case without colloidal silica.

[0155] [Table 7]

[0156]

[0157] [Table 8]

[0158]

[0159] (Saccharifying enzyme compositions of Examples 38-43)

[0160] First, an aqueous solution of cellulase is prepared as an aqueous solution of saccharifying enzyme through the following steps. Furthermore, the cellulase used is a cellulase derived from the genus *Trichoderma reesei* (prepared by Sigma Aldrich) that exhibits optimal enzymatic activity in the pH range of 3–6.

[0161] First, 0.5 g of cellulase powder was added to 9.5 g of deionized water and dissolved at room temperature using a magnetic stirrer for 2 hours while stirring, to obtain a 5% (w / w) aqueous solution of cellulase. Next, 20 g of alkaline silica sol (pH 9.0, silica concentration 40% (w / w)) obtained by dispersing solid spherical colloidal silica (average primary particle size: 35 nm, particle size determined by dynamic light scattering: 55 nm) in water was treated with a strong acidic hydrogen-form cation exchange resin, IR-120B (manufactured by Organo), to remove alkali metal ions, to obtain 20 g of acidic silica sol (pH 2.1, silica concentration 40% (w / w)). While stirring, deionized water and the aforementioned 5% (w / w) cellulase aqueous solution were added to the obtained acidic silica sol. Then, to adjust the pH, 0.05 M equivalents of one of the components in an acetate-Na acetate buffer solution (pH 4.0–6.0) were added to obtain a saccharifying enzyme composition with a silica concentration of 5% (w / w) and a cellulase concentration of 0.01–0.5% (w / w). The particle size of the obtained saccharifying enzyme composition was confirmed using dynamic light scattering, and the result was 55 nm, indicating no change in particle size within the colloidal silica dispersion.

[0162] Table 9 shows the saccharifying enzyme compositions of Examples 38–43.

[0163] (Comparative Examples 21-26: glucoamylase compositions)

[0164] Except that colloidal silica was not added, and deionized water was used instead to adjust the concentration of the saccharifying enzyme, the procedure was the same as in Examples 38-43. The saccharifying enzyme compositions are shown in Table 9.

[0165] The preparation of the saccharification reaction solution was carried out in the same manner as in Examples 1-8, except that the amount of microcrystalline cellulose powder was changed to 5.0% by mass. The method for producing sugar was carried out in the same manner as in Examples 1-8, except that the enzyme reaction period was changed to 7 days and the reaction temperature was changed to 40°C or 50°C. The determination of the saccharification reaction efficiency was carried out in the same manner as in Examples 1-8. The results of the saccharification rate determination are shown in Table 10.

[0166] In saccharification reaction solutions where the proportion of saccharifying enzymes not immobilized in colloidal silica to the total saccharifying enzymes is 25% to 100%, a high saccharification rate is observed in the range of 0.002 to 0.1 mass ratio of saccharifying enzymes to colloidal silica, which is also higher than that in the case without colloidal silica.

[0167] [Table 9]

[0168]

[0169] I: Sodium acetate buffer (pH = 6.0)

[0170] [Table 10]

[0171]

[0172] (The saccharifying enzyme composition of Example 44)

[0173] First, an aqueous solution of cellulase is prepared as an aqueous solution of saccharifying enzyme through the following steps. Furthermore, the cellulase used is a cellulase derived from the genus *Aspergillus niger* (prepared by MP biomedicals), which exhibits optimal enzymatic activity in the pH range of 3–6.

[0174] First, 2.0 g of cellulase powder was added to 38 g of deionized water and dissolved at room temperature using a magnetic stirrer for 2 hours while stirring to obtain a 5% (w / w) cellulase aqueous solution. Next, 12.0 g of deionized water and 2.0 g of the above-mentioned 5% (w / w) cellulase aqueous solution were added to 5.0 g of acidic silica sol (pH 2.9, silica concentration 20% (w / w)) obtained by dispersing solid spherical colloidal silica (average primary particle size: 45 nm, particle size determined by dynamic light scattering method: 75 nm) in water while stirring. Then, to adjust the pH, 1.0 g of 1 M equivalent of acetate-sodium acetate buffer (pH 4.0) was added to obtain a saccharifying enzyme composition with a silica concentration of 5% (w / w) and a cellulase concentration of 0.5% (w / w).

[0175] (The saccharifying enzyme composition of Example 45)

[0176] Acidic silica sol (pH 2.9, silica concentration 20% by mass) obtained by dispersing 5.0 g of solid spherical colloidal silica (average primary particle size: 45 nm, particle size measured by dynamic light scattering method: 75 nm) produced by the water glass method was added to water while stirring. This was followed by the addition of 12.0 g of deionized water, 7 ppm of aluminum chloride 83 (polyaluminum chloride, manufactured by Taimei Chemical Industry), and 2.0 g of the aforementioned 5% by mass cellulase aqueous solution. Then, to adjust the pH, 1.0 g of 1 M equivalent acetate-sodium acetate buffer (pH 4.0) was added to obtain a saccharifying enzyme composition with a silica concentration of 5% by mass and a cellulase concentration of 0.5% by mass.

[0177] (The saccharifying enzyme composition of Example 46)

[0178] Acidic silica sol (pH 2.9, silica concentration 20% by mass) obtained by dispersing 5.0 g of solid spherical colloidal silica (average primary particle size: 45 nm, particle size measured by dynamic light scattering method: 75 nm) produced by the water glass method was added to water while stirring. This was followed by the addition of 12.0 g of deionized water, 10 ppm of aluminum chloride 83 (polyaluminum chloride, manufactured by Taimei Chemical Industry), and 2.0 g of the aforementioned 5% by mass cellulase aqueous solution. To adjust the pH, 1.0 g of 1 M equivalent acetate-sodium acetate buffer (pH 4.0) was added to obtain a saccharifying enzyme composition with a silica concentration of 5% by mass and a cellulase concentration of 0.5% by mass.

[0179] (The saccharifying enzyme composition of Example 47)

[0180] Acidic silica sol (pH 2.9, silica concentration 20% by mass) obtained by dispersing 5.0 g of solid spherical colloidal silica (average primary particle size: 45 nm, particle size measured by dynamic light scattering method: 75 nm) produced by the water glass method was added to water while stirring. This was followed by the addition of 12.0 g of deionized water, 14 ppm of aluminum chloride 83 (polyaluminum chloride, manufactured by Taimei Chemical Industry), and 2.0 g of the aforementioned 5% by mass cellulase aqueous solution. Then, to adjust the pH, 1.0 g of 1 M equivalent acetate-sodium acetate buffer (pH 4.0) was added to obtain a saccharifying enzyme composition with a silica concentration of 5% by mass and a cellulase concentration of 0.5% by mass.

[0181] (The saccharifying enzyme composition of Example 48)

[0182] Acidic silica sol (pH 2.9, silica concentration 20% by mass) obtained by dispersing 5.0 g of solid spherical colloidal silica (average primary particle size: 45 nm, particle size measured by dynamic light scattering method: 75 nm) produced by the water glass method was added to water while stirring. This was followed by the addition of 12.0 g of deionized water, 15 ppm of aluminum chloride 83 (polyaluminum chloride, manufactured by Taimei Chemical Industry), and 2.0 g of the aforementioned 5% by mass cellulase aqueous solution. Then, to adjust the pH, 1.0 g of 1 M equivalent acetate-sodium acetate buffer (pH 4.0) was added to obtain a saccharifying enzyme composition with a silica concentration of 5% by mass and a cellulase concentration of 0.5% by mass.

[0183] (The saccharifying enzyme composition of Example 49)

[0184] Acidic silica sol (pH 2.9, silica concentration 20% by mass) obtained by dispersing 5.0 g of solid spherical colloidal silica (average primary particle size: 45 nm, particle size measured by dynamic light scattering method: 75 nm) produced by the water glass method was added to water while stirring. This was followed by the addition of 12.0 g of deionized water, 16 ppm of aluminum chloride 83 (polyaluminum chloride, manufactured by Taimei Chemical Industry), and 2.0 g of the aforementioned 5% by mass cellulase aqueous solution. Then, to adjust the pH, 1.0 g of 1 M equivalent acetate-sodium acetate buffer (pH 4.0) was added to obtain a saccharifying enzyme composition with a silica concentration of 5% by mass and a cellulase concentration of 0.5% by mass.

[0185] (Comparative Example 27: glucoamylase composition)

[0186] Acidic silica sol (pH 2.9, silica concentration 20% by mass) obtained by dispersing 5.0 g of solid spherical colloidal silica (average primary particle size: 45 nm, particle size measured by dynamic light scattering method: 75 nm) produced by the water glass method was added to water while stirring. This was followed by the addition of 12.0 g of deionized water, 20 ppm of aluminum chloride 83 (polyaluminum chloride, manufactured by Taimei Chemical Industry), and 2.0 g of the aforementioned 5% by mass cellulase aqueous solution. Then, to adjust the pH, 1.0 g of 1 M equivalent acetate-sodium acetate buffer (pH 4.0) was added to obtain a saccharifying enzyme composition with a silica concentration of 5% by mass and a cellulase concentration of 0.5% by mass.

[0187] (Comparative Example 28: Saccharifying enzyme composition)

[0188] Acidic silica sol (pH 2.9, silica concentration 20% by mass) obtained by dispersing 5.0 g of solid spherical colloidal silica (average primary particle size: 45 nm, particle size measured by dynamic light scattering method: 75 nm) produced by the water glass method was added to water while stirring. This was followed by the addition of 12.0 g of deionized water, 27 ppm of aluminum chloride 83 (polyaluminum chloride, manufactured by Taimei Chemical Industry), and 2.0 g of the aforementioned 5% by mass cellulase aqueous solution. To adjust the pH, 1.0 g of 1 M equivalent acetate-sodium acetate buffer (pH 4.0) was added to obtain a saccharifying enzyme composition with a silica concentration of 5% by mass and a cellulase concentration of 0.5% by mass.

[0189] (Comparative Example 29: Glycoamylase composition)

[0190] Except that colloidal silica was not added, and instead deionized water was used to adjust the concentration of the saccharifying enzyme, the process was carried out in the same manner as in Examples 44-49 to obtain a saccharifying enzyme composition that does not contain colloidal silica. This saccharifying enzyme composition is shown in Table 11.

[0191] Table 11 shows the saccharifying enzyme compositions of Examples 44–49.

[0192] The preparation of the saccharification reaction solution and the determination of the saccharification reaction efficiency were carried out in the same manner as in Examples 1-8. Regarding the sugar production method, it was carried out in the same manner as in Examples 1-8, except that the enzyme reaction period was changed to 7 days. The results of the saccharification rate determination are shown in Table 12. Furthermore, the dependence of the proportion of unimmobilized saccharifying enzymes on colloidal silica relative to the total saccharifying enzymes in the saccharification compositions of Examples 44-49 after 7 days of enzyme reaction is shown in Table 12. Figure 3 Given a constant mass ratio of saccharifying enzyme to colloidal silica (saccharifying enzyme / colloidal silica) and pH value, the saccharification rate depends on the proportion of saccharifying enzyme not immobilized in colloidal silica relative to the total saccharifying enzyme. A high saccharification rate is indicated when the proportion of saccharifying enzyme not immobilized in colloidal silica is between 27% and 77% relative to the total saccharifying enzyme.

[0193] [Table 11]

[0194]

[0195] [Table 12]

[0196]

[0197] (Comparative Example 30: Saccharifying enzyme composition)

[0198] First, 1 g of cellulase powder was added to 9 g of deionized water and dissolved at room temperature using a magnetic stirrer for 2 hours while stirring to obtain a 10% by mass cellulase aqueous solution. Next, 17.9 g of deionized water and 1.0 g of the above-mentioned 10% by mass cellulase aqueous solution were added to 0.1 g of sedimentation silica powder (trade name: Caprex #80, manufactured by DSL. Japan, average primary particle size: 7 nm, particle size measured by dynamic light scattering: 1750 nm) while stirring. Then, to adjust the pH value, 1.0 g of 1 M equivalent of acetate-sodium acetate buffer (pH 4.0) was added to obtain a saccharifying enzyme composition with a silica concentration of 5% by mass and a cellulase concentration of 0.5% by mass.

[0199] (Comparative Example 31: Glycoamylase composition)

[0200] While stirring, 17.9 g of deionized water and 1.0 g of the aforementioned 10% by mass cellulase aqueous solution were added to 0.1 g of sedimentation silica powder (trade name: Tokusil GU-N, manufactured by Tokuyama, average primary particle size: 11 nm, particle size measured by dynamic light scattering: 4740 nm). Then, to adjust the pH value, 1.0 g of 1 M equivalent of acetate-sodium acetate buffer (pH 4.0) was added to obtain a saccharifying enzyme composition with silica concentration of 5% by mass and cellulase concentration of 0.5% by mass.

[0201] Table 13 shows the saccharifying enzyme compositions of Comparative Examples 30 and 31.

[0202] The preparation of the saccharification reaction solution, the method of sugar production, and the determination of saccharification reaction efficiency were carried out in the same manner as in Examples 1-8. The results of the saccharification rate determination are shown in Table 14. Unlike colloidal silica, the saccharification rate was not improved when using silica powder obtained by the sedimentation method.

[0203] [Table 13]

[0204]

[0205] [Table 14]

[0206]

Claims

1. A saccharification reaction solution, characterized in that, It is a saccharification reaction solution in which at least one of cellulose and hemicellulose is saccharified, wherein the saccharifying enzyme and colloidal silica are contained in a dispersed state. The concentration of the saccharifying enzyme is 0.005% to 3.0% by mass. The concentration of the colloidal silica is 0.005% to 40% by mass. The mass ratio of the saccharifying enzyme to the colloidal silica, i.e., the saccharifying enzyme / colloidal silica ratio, is 0.002 to 300. Furthermore, the average primary particle size of the colloidal silica is 1 nm to 400 nm, and the particle size measured by dynamic light scattering method is greater than or equal to 5 nm and less than 500 nm. The proportion of saccharifying enzymes not immobilized in colloidal silica relative to all saccharifying enzymes is 50%–100%. The pH value of the saccharification reaction solution is 3 to 7.

2. The saccharification reaction solution according to claim 1, characterized in that, The saccharifying enzyme comprises at least one of a saccharifying enzyme derived from the genus Aspergillus and a saccharifying enzyme derived from the genus Trichoderma.

3. A saccharifying enzyme composition, characterized in that, It is a saccharification composition in which at least one of cellulose and hemicellulose is saccharified, wherein the saccharifying enzyme and colloidal silica in a dispersed state have an average primary particle size of 1 nm to 400 nm and a particle size greater than or equal to 5 nm and less than 500 nm as determined by dynamic light scattering, and the proportion of the saccharifying enzyme not immobilized in the colloidal silica to the total saccharifying enzyme is 50% to 100%.

4. A method for manufacturing sugar, characterized in that, Sugar is produced using the saccharification reaction solution as described in claim 1 or 2.

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