JP2009203454A - Method of producing cellulose decomposition product utilizing ionic liquid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cellulose treatment method for efficiently decomposing by cellulase the cellulose solubilized with an ionic liquid. <P>SOLUTION: An ionic liquid in which the cellulose in a cellulose-containing material is solubilized is prepared, a predetermined amount of a poor solvent of the cellulose in the relation to the ionic liquid is added to the above ionic liquid containing the solubilized cellulose, and a cellulase acting environment is formed which contains the above ionic liquid and the above solvent and in which cellulase can offer enzyme activity. This makes it possible to impart, to the cellulose after the solubilization by the ionic liquid, an environment in which the cellulase can effectively act on the cellulose by a simple operation or in a less amount of use of the cellulase. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a method for treating a cellulose-containing material using an ionic liquid, a method for producing a decomposition product, an enzyme reaction medium, and the like.

  As an alternative to finite petroleum resources, there is an increasing expectation for biomass derived from the photosynthetic action of plants, and various attempts have been made to use biomass for energy and various materials. In order to effectively use biomass as an energy source and other raw materials, it is necessary to saccharify biomass into a carbon source that can be easily used by animals and microorganisms. In addition, the importance of biorefinery, which is an attempt to use biomass for chemical products and biofuels, has been pointed out, and technological development for practical application is underway. Problems to be solved for practical use include the development of an efficient decomposition method for cellulose, which is the main component of woody or herbaceous biomass, particularly crystalline cellulose constituting the crystal structure.

  In the current saccharification process, biomass is pretreated by high temperature / high pressure treatment or acid treatment, and then cellulase is allowed to act. However, since a large amount of energy is required for pretreatment and a large amount of cellulase is required, it is a big problem in practical use.

  In recent years, it has been reported that ionic liquids solubilize cellulose. For example, it has been found that cellulose is solubilized in a chloride-based ionic liquid at about 100 ° C. (Patent Document 1, Non-Patent Document 1). It has also been found that non-chloride ionic liquids can solubilize cellulose under milder conditions (Patent Document 2, Non-Patent Documents 2, 3, and 4).

  Furthermore, attempts have been made to saccharify cellulose solubilized with ionic liquid with cellulase, but it has been reported that cellulase is inactivated in ionic liquid (Non-patent Documents 2 and 4). After pretreatment of solubilizing cellulose with an ionic liquid, it is reported that the solubilized cellulose can be washed with a poor solvent such as water to remove the ionic liquid, and then put into water to decompose with cellulase. (Non-Patent Document 5).

JP 2005-506401 A JP 2006-137777 A R D. Roger et al., J. Am. Chem. Soc. 124 (18), 4974-4975, 2002 Ohno et al., Polym. Prep. Jpn., 55 (1), 2090, 2006 Ohno et al., Polym. Prep. Jpn., 56 (1), 2198-2199, 2007 R D. Roger et al., Green Chem., 5, 443-447, 2003 C A. Schall et al., Biotechnol. Bioeng., 95 (5), 904-910, 2006

  However, Non-Patent Documents 2 and 4 suggest that it may be effective to stabilize cellulase with polyethylene oxide (PEO) or the like in order to decompose cellulose solubilized with ionic liquid with cellulase. However, cellulolytic activity sufficient for the stabilized cellulase obtained at present is not obtained. In addition, as described in Non-Patent Document 5, when cellulose is solubilized with an ionic liquid, a careful washing operation for removing the ionic liquid from cellulose is required, and still a considerably large amount of cellulase is required. I understood it. In addition, the fact that cellulose must be decomposed and saccharified in a reaction field different from that obtained by solubilizing cellulose with an ionic liquid is inconvenient for efficient cellulose decomposition and utilization.

  As described above, at present, cellulose can be solubilized with an ionic liquid, but it has still been difficult to efficiently decompose it with cellulase. Further, it is required to efficiently decompose cellulose using an ionic liquid and to stably decompose cellulose at a certain decomposition efficiency.

  An object of the present invention is to provide a cellulose treatment method for efficiently decomposing cellulose solubilized with an ionic liquid with cellulase. More specifically, the present invention provides a treatment method capable of imparting an environment in which cellulase can effectively act on cellulose in the same reaction field or with a smaller amount of cellulase used, to cellulose solubilized with an ionic liquid. One purpose. Another object of the present invention is to provide a method for producing a cellulose degradation product capable of efficiently degrading cellulose solubilized with an ionic liquid with cellulase to obtain a cellulose degradation product. Another object of the present invention is to provide a cellulose-decomposing medium suitable for degrading and saccharifying cellulose solubilized with an ionic liquid with cellulase. Another object of the present invention is to provide an enzyme reaction medium suitable for solubilizing a poorly soluble substrate as an enzyme substrate.

  The inventors of the present invention have intensively studied to solve the above-described problems, and once solubilized with an ionic liquid, the presence of the ionic liquid is obtained by allowing cellulase to act in a state where a poor solvent or an antisolvent is added. It was found that cellulose can be effectively decomposed by cellulase even under the above conditions. Furthermore, the present inventors diligently studied the decomposition activity of cellulose using various ionic liquids. As a result, even when the same operation was performed using the same type of ionic liquid, there was a large variation in the decomposition efficiency of cellulose. As well as the knowledge that the problem can be resolved. Based on these findings, the inventors have created the present invention. That is, according to the present invention, the following means for solving at least one of the above-described problems are provided.

  According to the present invention, there is provided a method for treating a cellulose-containing material, the step of preparing an ionic liquid in which cellulose in the cellulose-containing material is solubilized, and the ionic liquid containing the solubilized cellulose in the ionic liquid, Adding a predetermined amount of a poor solvent in relation to the ionic liquid with respect to cellulose, and forming a cellulase working environment that includes the ionic liquid and the poor solvent so that the cellulase can exhibit enzyme activity. Provided.

  In the treatment method, at least a part of cellulose may be precipitated in the cellulase working environment. Further, in the cellulase working environment forming step or prior to the step, the poor solvent may be added to the ionic liquid containing the solubilized cellulose to precipitate the cellulose.

  According to the present invention, there is provided a method for treating a cellulose-containing material, the step of preparing an ionic liquid in which cellulose in the cellulose-containing material is solubilized, and the ionic liquid containing the solubilized cellulose in the ionic liquid, Adding a poor solvent in relation to the ionic liquid with respect to cellulose, and forming a cellulase working environment that includes the ionic liquid and the poor solvent so that cellulase can exhibit enzyme activity, and as the poor solvent, A treatment method is provided that includes controlling the pH of the cellulase working environment.

  In the method, when the ionic liquid is mixed with the poor solvent, a poor solvent having an adjustment ability to correct a shift from a preferred pH preferable for the cellulase to act or an adjustment ability to suppress pH fluctuation with respect to the preferred pH. It is preferable to use it. In addition, the ionic liquid is preferably an ionic liquid containing a cation selected from a carboxylic acid anion, a halogen anion, and a phosphate anion, and an imidazolium cation. Furthermore, the anion may be a halogen-based anion.

  According to the present invention, there is provided a method for producing a cellulose degradation product, the step of preparing an ionic liquid in which cellulose in the cellulose-containing material is solubilized, and the ionic liquid containing the solubilized cellulose in the ionic liquid, Adding a predetermined amount of a poor solvent in relation to the ionic liquid in relation to cellulose, forming a cellulase working environment that includes the ionic liquid and the poor solvent and in which cellulase can exhibit enzyme activity; and the cellulase containing the cellulase And a step of decomposing cellulose under an operating environment.

  According to the method for producing a cellulose degradation product of the present invention, it is possible to form an environment suitable for degrading cellulose solubilized by an ionic liquid with cellulase and saccharifying it. It can be degraded and saccharified.

  In the method for producing a cellulose degradation product of the present invention, the cellulase working environment may be one in which at least a part of cellulose is deposited, and in this aspect, in the cellulase working environment forming step or in the step. Prior to this, the poor solvent may be added to the ionic liquid containing the solubilized cellulose to precipitate the cellulose.

  In the method for producing a cellulose degradation product of the present invention, the cellulase working environment may contain the poor solvent 1.5 times or more in a volume ratio with respect to the ionic liquid. The solvent may be an aqueous medium. Preferably, the poor solvent is water or a buffer solution, and more preferably a buffer solution. Further, the poor solvent may contain cellulase.

  In the method for producing a cellulose degradation product of the present invention, the ionic liquid is preferably non-halogen. The ionic liquid preferably contains a phosphate anion or a carboxylic acid anion. Furthermore, the ionic liquid preferably contains 1-ethyl-3-methylimidazolium cation.

  In the method for producing a cellulose degradation product of the present invention, the cellulase preferably contains a cellulase derived from the genus Trichoderma or Aspergillus.

  Furthermore, in the method for producing a cellulose degradation product of the present invention, the step of preparing an ionic liquid in which cellulose in a cellulose-containing material is solubilized, and the ionic liquid containing the solubilized cellulose in the ionic liquid, the cellulose Adding a poor solvent in relation to an ionic liquid, including the ionic liquid and the poor solvent, forming a cellulase working environment in which cellulase can exhibit enzyme activity, and under the cellulase working environment containing the cellulase, A step of decomposing cellulose, and when the ionic liquid is mixed with the poor solvent as the poor solvent, the adjustment ability to correct a shift from a preferred pH preferred for the cellulase to act or the preferred pH A production method is provided that uses a poor solvent having an adjustment ability to suppress pH fluctuations.

  In this aspect, the ionic liquid may be an ionic liquid having an anion selected from a carboxylic acid anion, a halogen anion, and a phosphate anion and an imidazolium cation as constituent ions. The anion may be a halogen anion.

  According to the present invention, there is provided a method for saccharification of cellulose, the step of preparing an ionic liquid in which cellulose in the cellulose-containing material is solubilized, and the ionic liquid containing the solubilized cellulose in relation to the cellulose. A saccharification method comprising: adding a predetermined amount of a poor solvent in relation to the ionic liquid, and forming a cellulase working environment including the ionic liquid and the poor solvent and allowing cellulase to exhibit enzyme activity. The

  In the cellulose saccharification method of the present invention, a step of preparing an ionic liquid in which cellulose in the cellulose-containing material is solubilized, and the ionic liquid containing the solubilized cellulose in the ionic liquid with respect to the cellulose A step of adding a poor solvent in the relationship to form a cellulase working environment in which cellulase can exhibit enzyme activity, including the ionic liquid and the poor solvent, wherein the ionic liquid is used as the poor solvent. When mixed with a saccharification method, a saccharification method using an antisolvent having an adjustment ability for correcting a shift from a preferred pH preferable for the cellulase to act or an adjustment ability for suppressing pH fluctuation with respect to the preferred pH is provided.

According to the present invention, an ionic liquid capable of solubilizing cellulose, which is a cellulose decomposition medium,
A decomposition medium comprising a poor solvent in relation to the ionic liquid with respect to the cellulose, and an amount of the solvent capable of forming a cellulase working environment in which the cellulase can exhibit enzyme activity in the presence of the ionic liquid. Provided. The medium of the present invention may further contain cellulose at least once solubilized with the ionic liquid. In this embodiment, at least a part of the cellulose may be precipitated. Furthermore, the cellulose decomposition medium of the present invention may contain cellulase.

  According to the present invention, the enzyme reaction medium is an ionic liquid capable of solubilizing a substrate, and the substrate is a poor solvent in relation to the ionic liquid, and the enzyme is mixed with the ionic liquid in the presence of the ionic liquid. There is provided a reaction medium comprising an amount of a solvent capable of forming an enzyme working environment capable of exerting enzyme activity.

  The present invention relates to a method for treating a cellulose-containing material, a method for producing a cellulose degradation product, and a composition for cellulose degradation. Hereinafter, these embodiments of the present invention will be described in detail with appropriate reference to the drawings. FIG. 1 shows an example of a method for treating cellulose according to the present invention, and FIG. 2 shows an example of a method for producing a cellulose degradation product according to the present invention.

(Method for treating cellulose-containing material)
As shown in FIG. 1, the processing method of the cellulose containing material of this invention is equipped with the preparation process of the ionic liquid which solubilized the cellulose, and the cellulase action environment formation process.

(Cellulose-containing material)
In the present specification, cellulose refers to a polymer in which glucose is polymerized by β-1,4-glucoside bonds and derivatives thereof. The degree of polymerization of glucose in cellulose is not particularly limited, but is preferably 200 or more. In addition, examples of the derivative include derivatives such as carboxymethylation, aldehyde formation, or esterification. Cellulose may contain cellooligosaccharide and cellobiose, which are partially decomposed products thereof. Further, the cellulose may be a complex with β-glucoside, which is a glycoside, lignocellulose, which is a complex with lignin and / or hemicellulose, and pectin. The cellulose may be crystalline cellulose or non-crystalline cellulose, but preferably contains crystalline cellulose. Furthermore, the cellulose may be naturally derived or artificially synthesized. The origin of cellulose is not particularly limited. It may be derived from plants, fungi, or bacteria.

  Cellulose exists in nature as a major component of plant cell walls, and is the most produced polysaccharide on the earth. In the plant cell wall, the cellulose forms a crystalline cellulose region and an amorphous cellulose region. The crystalline cellulose region forms a strong crystal structure due to intermolecular hydrogen bonding or the like, and requires extremely harsh conditions to be artificially decomposed into monosaccharides such as glucose. In nature, cellulose is degraded by microorganisms such as filamentous fungi, but is known to be degraded to glucose by a synergistic action of various cellulases.

  In the present specification, the cellulose-containing material may be any material that contains cellulose as described above. Cellulose-containing materials include natural fiber products such as cotton and linen, recycled fiber products such as rayon, cupra, acetate, and lyocell, various straws such as rice straw, agricultural waste such as rice husks, bagasse and wood chips, waste paper, and building waste. Biomass (woody and herbaceous) containing various wastes such as

(Preparation process of ionic liquid solubilized cellulose)
The preparation step of the ionic liquid solubilized cellulose is a step of preparing an ionic liquid in which the cellulose in the cellulose-containing material is solubilized. The ionic liquid in which cellulose is solubilized may be obtained by itself, or may be obtained by solubilizing cellulose of a cellulose-containing material with an ionic liquid. The ionic liquid for solubilizing cellulose is not particularly limited, and various ionic liquids can be used. According to the present invention, neither a cation nor an anion is a non-halogen ionic liquid containing no halogen, but an ionic liquid containing a halogen anion or the like. In addition, the ionic liquid is preferably an ionic liquid having an anionic species with high hydrogen bond acceptability. This is because such an ionic liquid tends to solubilize cellulose. Moreover, such an ionic liquid is compatible with a medium in which cellulase acts, such as water or a buffer solution.

  Although it does not specifically limit as a cation which comprises an ionic liquid, A heterocyclic onium cation and an ammonium cation are mentioned. Examples of the heterocyclic onium cation include an imidazolium cation represented by the following “Chemical Formula 1”, a pyridinium cation represented by “Chemical Formula 2”, and the like.

(However, R ¹ and R ² each independently represents an alkyl group.)

In “Chemical Formula 1” and “Chemical Formula 2”, examples of the alkyl group in R ¹ and R ² include a methyl group, an ethyl group, a propyl group, an isopropyl group, a butyl group, an isobutyl group, a sec-butyl group, and a tert-butyl group. A pentyl group, an isopentyl group, a hexyl group, a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, and a cyclohexyl group. More preferably, it is a C1-C6, More preferably, it is a C1-C4 alkyl group. The alkyl group may be linear, branched or cyclic, but is preferably linear. Carbon atoms of the imidazole ring of the imidazolium cation represented by “Chemical Formula 1” and the pyridine ring of the pyridinium cation represented by “Chemical Formula 2” may be substituted. R ¹ and R ² in “Chemical Formula 1” may be a methyl group and an ethyl group, a methyl group and a butyl group, an ethyl group and a butyl group, and the like.

  Specific examples of such imidazolium cations include 1-ethyl-3-methylimidazolium ion, 1-butyl-3-methylimidazolium ion, 1-propyl-3-methylimidazolium ion, and 1-pentyl-3. -Methylimidazolium ion, 1-hexyl-3-methylimidazolium ion, 1-heptyl-3-methylimidazolium ion, 1-octyl-3-methylimidazolium ion, 1-nonethyl-3-methylimidazolium ion, Dialkylimidazolium cations such as 1-decyl-3-methylimidazolium ion, 1- (1,2 or 3-hydroxypropyl) -3-methylimidazolium ion, 1,2,3-trimethylimidazolium ion, 1, 2-Dimethyl-3-propylimidazolium ion Trialkylimidazolium cation such as 1-butyl-2,3-dimethyl imidazolium ion. Of these, dialkylimidazolium cations such as 1-ethyl-3-methylimidazolium cation, 1-butyl-3-methylimidazolium ion, and 1-propyl-3-methylimidazolium ion are preferable.

  Examples of the pyridinium cation include N-propylpyridinium ion, N-butylpyridinium ion, 1-butyl-4-methylpyridinium ion, 1-butyl-2,4-dimethylpyridinium ion and the like.

  Examples of the ammonium cation include trimethylpropylammonium ion, trimethylhexylammonium ion, tetrapentylammonium ion, aliphatic quaternary ammonium ion such as diethyltrimethyl (2-methoxyethyl) ammonium ion, N-butyl-N-methylpyrrole, and the like. Examples include alicyclic quaternary ammonium ions such as dinium ions.

Examples of the anion constituting the ionic liquid is not particularly limited, for ^{_{example, HSO 4 -, CH 3 SO}} 3 - can also be used inorganic anions such. Carboxylic acid anions are also preferable, and among them, C1-C4 carboxylic acid anions such as formic acid anions, acetic acid anions, and propionic acid anions are more preferable. More preferred is an acetate anion.

  In addition, the anion may be various halogen anions such as chlorine ion, bromine ion and iodine ion. In particular, according to the present invention, even in the case of a halogen anion ionic liquid, by adjusting the pH of the cellulase working environment with a poor solvent having a high buffering capacity, the inactivation of the enzyme can be suppressed and the efficiency can be stably increased. Cellulose can be decomposed.

  In view of the mildness of cellulose solubilization conditions and the effect on cellulase, phosphate anions and carboxylic acid anions are preferred. More preferably, it is a phosphate anion. Examples of the phosphate anion include a phosphate anion represented by the following “Chemical Formula 3”.

(However, R ⁴ and R ⁵ each independently represents an alkyl group.)

The alkyl group in R ⁴ and R ⁵ of “Chemical Formula 3” has the same meaning as the alkyl group in R ¹ , R ² and R ³ of “Chemical Formula 1” and “Chemical Formula 2”. R ⁴ and R ⁵ in “Chemical Formula 3” are preferably each independently a methyl group or an ethyl group. Specific examples of such phosphate anions include (CH ₃ O) ₂ PO ₂ ⁻ , (CH ₃ O) CH ₃ PO ₂ ⁻ , (CH ₃ O) C ₂ H ₅ OPO ₂ ⁻ and (CH ₃ O ) HPO ₂ ⁻ , (C ₂ H ₅ O) ₂ PO ₂ ⁻ , (C ₂ H ₅ O) CH ₃ PO ₂ ⁻ , (C ₂ H ₅ O) C ₂ H ₅ PO ₂ ⁻ , (C ₂ H ₅ O) HPO ₂ ^- and the like.

  Carboxylic acid anions are also preferable, and among them, C1-C4 carboxylic acid anions such as formic acid anion, acetic acid anion, and propionic acid anion are more preferable. More preferred is an acetate anion.

  Preferred examples of the ionic liquid include 1-ethyl-3-methylimidazolium diethyl phosphate or 1-ethyl-3-methylimidazolium acetic acid, 1-ethyl-3-methylimidazolium dimethyl phosphate, 1-ethyl-3 -Methylimidazolium hexafluorophosphoric acid, 1-ethyl-3-methylimidazolium diethylphosphoric acid tetrafluoroboric acid, 1-ethyl-3-methylimidazolium phosphoric acid, 1-ethyl-3-methylimidazolium methylsulfuric acid, Examples include 1-ethyl-3-methylimidazolium dimethyl sulfate and 1-ethyl-3-methylimidazolium ethyl sulfate.

  Preferred ionic liquids include, for example, 1-ethyl-3-methylimidazolium chlorine, 1-ethyl-3-methylimidazolium bromine, 1-ethyl-3-methylimidazolium iodine, 1-butyl-3-methyl. Imidazolium chlorine, 1-butyl-3-methylimidazolium bromine, 1-butyl-3-methylimidazolium iodine, 1-octyl-3-methylimidazolium chlorine, 1-octyl-3-methylimidazolium bromine, 1- Examples include octyl-3-methylimidazolium iodine.

  The ionic liquid can be synthesized by a known method except that it is commercially available. The synthesis method is not particularly limited, and the cation is synthesized and purified as a salt with chloride, and then reacted with the anion salt of the ionic liquid to be obtained, or once converted into a hydroxide, You may neutralize with the acid which contains.

  The purity of the ionic liquid is preferably high. This is because impurities in the process of synthesizing the ionic liquid greatly affect the pH of the medium obtained by mixing the ionic liquid and the poor solvent, and consequently greatly affect the catalytic activity of the enzyme. According to the present inventors, in the case of an ionic liquid using an imidazolium-based cation, the lower the purity, the easier the pH when mixed with a poor solvent such as water or a buffer solution is shifted to an alkali, and cellulolytic activity by cellulase is increased. It has been found that there is a tendency to decline.

  Therefore, it is preferable to use an ionic liquid with high purity, but more specifically, an ionic liquid suitable for forming a cellulase working environment is selected, or pH is adjusted with an appropriate strength, and then the ionic liquid is selected. Is preferably used. When selecting an ionic liquid or selecting a pH adjustment strength for forming a cellulase working environment, the pH when mixed at a predetermined ratio with a predetermined poor solvent such as water or a predetermined buffer is used as an index. Can do. For example, a buffer (poor solvent) having a pH in the vicinity of the optimum pH of cellulase such as 10 mM citrate buffer (pH 5.0) and a certain ratio, for example, ionic liquid: poor solvent = 1: 1.5 to 1: Mixing in the range of about 4 and measuring the pH, cellulase working environment can be formed with the ionic liquid and a poor solvent as it is when the optimum pH of cellulase or its vicinity, for example, pH 6 or less. On the other hand, if the pH at the time of mixing exceeds the optimum pH of cellulase or in the vicinity thereof, for example, pH 6, the ionic liquid is not selected, or the ionic liquid has a higher pH adjustment ability (or is adjusted to be more acidic). A cellulase working environment may be formed using a buffer solution having a function.

  One type of ionic liquid may be used, or two or more types may be used. Moreover, it is preferable that the melting | fusing point of an ionic liquid is 100 degrees C or less, More preferably, it is 80 degrees C or less. More preferably, it is 40 degrees C or less, Most preferably, it is 20 degrees C or less. Further, from the viewpoint of dissolving cellulose, the ionic liquid preferably has a water content of several mass% or less, more preferably 3 mass% or less, and even more preferably 1 mass% or less. The amount of water can be measured by the Karl Fischer method.

  The method for solubilizing cellulose with an ionic liquid is not particularly limited. It can be realized by adding cellulose to the ionic liquid and stirring as necessary. When the ionic liquid is dissolved with cellulose, it can be performed at room temperature, and heating of the ionic liquid is not necessarily required. However, the ionic liquid can be heated as necessary. Although heating temperature is not specifically limited, What is necessary is just to heat so that it may become about the melting temperature of the ionic liquid acquired previously. Whether cellulose is dissolved in the ionic liquid can be easily determined by the fact that the cellulose is dispersed and the ionic liquid that is clogged becomes clear. The concentration of cellulose solubilized in the ionic liquid is not particularly limited. Usually, it is about 10% by mass or less, and typically about 5% by mass or less.

(Cellulase working environment formation process)
In this step, a predetermined solvent is added to the ionic liquid containing the solubilized cellulose to form a cellulase working environment containing the ionic liquid and the solvent and allowing the cellulase to exert its enzymatic activity. According to this step, a cellulase working environment in which cellulose can be decomposed and saccharified with cellulase can be formed by adding a predetermined solvent to the ionic liquid containing solubilized cellulose. That is, it is possible to decompose and saccharify with cellulase in the solubilized spot without washing the precipitated cellulose with water or the like, or transferring it to a separately prepared enzyme reaction medium. In addition, when the ionic liquid coexists in the reaction field of cellulase, cellulose takes a form that cellulase is likely to attack, and cellulase can efficiently decompose cellulose.

  The cellulase working environment refers to an environment in which cellulase can exhibit the enzyme activity of cellulase with respect to cellulose. In any environment where ionic liquid and poor solvent exist, cellulase can be operated in combination with ionic liquid solubilized cellulose and poor solvent at various blending ratios. By evaluating the degree of cellulose degradation (cellulase activity) by cellulase, it is possible to confirm which liquid is in an environment in which cellulase can operate.

  Cellulase activity can be evaluated by a known method. For example, it can be evaluated by measuring the amount of reducing sugar produced by the decomposition of cellulose. Methods for quantifying reducing sugars include the Somogyi method, the Tauber-Kleiner method, the Hanes method (titration method), the Park-Johnson method, the 3,5-dinitrosalicylic acid (DNS) method, and the TZ method (Journal of Biochemical Methods, 11 (1985). 109-115), phenol-sulfuric acid method (absorbance method), lane-einone (titration method) and many other quantitative methods. In addition, the Somogyi-Nelson method using the reduction of copper ions by sugar can be used (Sakuzo Fukui, “Biochemical Experimental Method 1 Quantitative Method for Reducing Sugar, Second Edition”, Society of Science Publishing Center 1990). Alternatively, ABEE (ABEE: 4-aminobenzoic acid ethyl ester) (Yasuno et al., Biosci. Biotech. Biochem. 61, 1994-1946) may be used for quantitative determination by HPLC or the like.

  Cellulase is a general term for various enzymes that act to hydrolyze cellulose to glucose. As cellulases, β1,4-endoglucanase (EC 3.2.1.4), glucan 1,4-β glucosidase (EC 3.2.1.74), cellulose 1,4-β cellobiosidase (EC 3) are narrowly defined. 2.1.91), β-glucosidase (EC 3.2.1.21). The cellulase may be naturally derived or artificially modified. Although it does not specifically limit as a thing of natural origin, The cellulase derived from the genus Trichoderma or Aspergillus etc. can be used preferably. In the present invention, the above-mentioned narrowly defined cellulases can be used singly or in combination of two or more. Instead of different types of cellulases, they may be the same type or a combination of two or more types. In addition, cellulases having different origins can be used in combination.

  The solvent added to the ionic liquid containing the solubilized cellulose is a poor solvent in relation to the ionic liquid used for cellulose. That is, it is a solvent in which cellulose is less soluble than the ionic liquid used. Such a solvent is usually a solvent other than a non-ionic liquid due to the nature of the ionic liquid, and specifically, an aqueous medium or an organic solvent. In view of enzyme activity and the like, an aqueous medium is preferably used. The aqueous medium includes water, an aqueous solution (such as a buffer solution), and a mixture of these and an organic solvent. In view of the enzyme reaction, the solvent is preferably water or a buffer. A buffer solution is more preferable. The pH of the buffer solution can be determined in consideration of the optimum pH of the enzyme to be used, that is, cellulase, and the details will be described later. One kind of poor solvent may be used, or two or more kinds may be used in combination.

(Control of pH in cellulase working environment)
In the present invention, an ionic liquid containing solubilized cellulose and a poor solvent form a cellulase working environment in which cellulase acts on cellulose, that is, a medium for decomposing cellulose by cellulase. In forming this medium, the pH of the medium is controlled. The ionic liquid itself is a salt, but depending on the impurities at the time of synthesis of the ionic liquid and the types and ratios of the anion and cation of the ionic liquid, the acid / basicity and pH in the ionic liquid aqueous solution may be different from the same ionic species. Even an ionic liquid becomes different. It has not been known at the time of filing this application how much such a difference affects the enzyme activity of cellulase in a mixed medium of a poor solvent such as water and an ionic liquid. The present inventors have found that such a difference in characteristics in the ionic liquid used is extremely important for the cellulase reaction in the ionic liquid-antisolvent medium, and have completed the present invention.

  In order to control the pH of the cellulase working environment, depending on the pH of the solution obtained by mixing the ionic liquid and the poor solvent, a preferred pH (preferable pH, more preferably optimum) for the cellulase used to act on cellulose. pH) is preferably adjusted. For such pH adjustment, adjustment ability to correct the shift from the preferred pH (typically optimum pH) preferred for cellulase to act as a poor solvent, or adjustment to suppress pH fluctuation relative to the preferred pH It is preferable to use a poor solvent having ability.

  Examples of such a poor solvent include the following. For example, when the pH of a mixed solution obtained by mixing an ionic liquid and a poor solvent is an ionic liquid that is on the alkali side of the optimum pH, a more preferable pH (more acidic side pH) when mixed It is possible to use a poor solvent capable of adjusting such that the reaction medium is obtained. Such an anti-solvent can be obtained by, for example, increasing the concentration of acid solution and salt with an aqueous solution that is more acidic than the optimum pH of cellulase, the ability to adjust to the acidic pH more than the optimum pH, and / or the buffering effect. A buffer solution having an ability to adjust to the acidic pH can be used as a poor solvent.

  Conversely, when the ionic liquid is such that the pH of the mixture obtained by mixing the ionic liquid and the poor solvent is more acidic than the optimum pH, for example, an aqueous solution that is more alkaline than the optimum pH of cellulase. The buffer solution having the ability to adjust to the alkaline side pH is used as a poor solvent by increasing the concentration of the acid and salt having a buffering action and / or the ability to adjust to the alkaline side pH from the optimum pH. Can do.

  In consideration of the stability of the enzyme reaction and the ease of operation, it is preferable to use a buffer as a poor solvent. It is preferable to use a buffer solution having a buffer capacity in a range corresponding to the pH range suitable for the enzyme activity including the optimum pH of the cellulase to be used. Since typical preferred pH of general cellulase is about 4 to 6, for example, citrate buffer (citric acid and sodium citrate), acetate buffer (acetic acid-sodium acetate), citrate-phosphate buffer Liquid (citric acid-sodium dihydrogen phosphate) and the like. It is preferable to appropriately adjust the concentration and pH of such a buffer so that the pH of the cellulase working environment is 4 or more and 6 or less, and more reliably 4.0 or more and 6.0 or less.

  In order to suppress fluctuations in the cellulase working environment due to fluctuations in the characteristics of the ionic liquid, it is effective to form a cellulase working environment at a suitable pH of the cellulase, preferably near the optimum pH, by adjusting to a more acidic pH. is there. According to the present inventors, when the ionic liquid is dissolved in a poor solvent such as water, the dissolved liquid has a high tendency to exhibit alkalinity. For such pH adjustment, as described above, it is preferable to use, as a poor solvent, a buffer solution having a larger buffering capacity to acidic pH.

  As a preferable buffer solution, a buffer solution having a concentration of about 10 to 100 times the normal reagent (acid and conjugate base) concentration, for example, the optimum pH of cellulase to be used is 5.0 and pH 5.0. When the above buffer solution is used, a buffer solution having a concentration of about 100 mM to 1 M, more preferably, a buffer solution having a concentration of about 200 mM to 600 mM can be used. Further, for example, when the optimum pH of the cellulase to be used is 5.0 and a buffer solution having a concentration of 10 mM is used, a buffer solution having a pH lower than the optimum pH, for example, pH 4.0 may be used. it can.

  The pH of the cellulase working environment may be controlled by controlling the characteristics of the ionic liquid used. For example, the pH when the ionic liquid is dissolved in water at a constant concentration may be measured, and the pH may be within a certain range. In order to regulate such pH, it can be preferably used by forming a cellulase working environment using each ionic liquid constituting an ionic liquid aqueous solution of various pHs, and measuring the pH, the enzyme activity of cellulase, and the like. The range of the ionic liquid properties (pH of the ionic liquid aqueous solution) can be determined.

  The pH control of the cellulase working environment is useful for ionic liquids that combine various anions and cations already described. That is, it is useful for ionic liquids having various anions selected from carboxylic acid anions, halogen anions and phosphate anions as constituent anions, but is particularly preferably used for ionic liquids having halogen anions as constituent anions. be able to. This is because conventionally this type of ionic liquid has good solubility of cellulose. In addition, it is thought that the enzyme activity of a cellulase is inhibited in the ionic liquid which uses a halogen-type anion as a constituent anion, and the enzyme reaction of a cellulase advances in the presence of the said ionic liquid. It can be said that even those skilled in the art could not have predicted.

  When considering the pH control of the cellulase working environment, it is useful for ionic liquids having an imidazolium cation such as alkylimidazolium as a cation. This is because this type of ionic liquid is widely used and has good solubility in cellulose. This is because this kind of ionic liquid is considered to contain an imidazole derivative as an impurity, and has a strong tendency to exhibit alkalinity when mixed with a poor solvent such as water.

  In addition, the poor solvent should just be supplied to an ionic liquid to such an extent that a cellulase can exhibit the enzyme activity. That is, the amount of the poor solvent added to the ionic liquid may be an amount that can form the obtained cellulase working environment. The amount of such an anti-solvent is mainly determined by the type of ionic liquid, the degree of characteristics that may inhibit the enzyme reaction of the ionic liquid, the type of cellulase and the type of anti-solvent (type of salt, strength of buffer capacity, etc. ). One of ordinary skill in the art can determine the appropriate amount of solvent based on the disclosure herein. For example, after solubilizing cellulose in an ionic liquid, a poor solvent to be used at a different mixing ratio is added to the ionic liquid containing the solubilized cellulose, and the degree of cellulose degradation by cellulase is evaluated for each of these mixed liquids By doing so, an appropriate blending ratio, that is, a predetermined amount of the solvent to be added can be obtained.

  Although it varies depending on the case, it is preferable to use a poor solvent (preferably an aqueous medium, more preferably water or a buffer solution) of approximately the same amount or more, preferably 1.5 times or more, by volume ratio with respect to the ionic liquid. . This is because if it is less than 1.5 times, it is difficult to obtain the effect of solubilizing cellulose with an ionic liquid. More preferably, it is 2 times or more, more preferably 3 times or more, and still more preferably 4 times or more. On the other hand, it is preferably 10 times or less from the viewpoint of process efficiency and cellulose dissolution.

  In the cellulase working environment, the cellulose need not be dissolved, and at least a part of the cellulose may be precipitated. This is because the cellulase working environment is such that a poor solvent is added to the ionic liquid solubilized with cellulose to such an extent that the cellulase can operate, and the solubility of cellulose is lowered in this medium. On the other hand, since the ionic liquid coexists, even if the cellulose is precipitated, the crystal structure obtained when it is solubilized with the ionic liquid can be retained to some extent or partially. Yes (see Non-Patent Document 1). Therefore, in the cellulase working environment, part or all of cellulose may be precipitated.

  When at least a part of cellulose is deposited in the cellulase working environment, the cellulose may be deposited at the same time as the poor solvent is added to form the cellulase working environment. Prior to forming the cellulase working environment, a poor solvent may be added to the ionic liquid containing solubilized cellulose to precipitate the cellulose. That is, a large amount of a poor solvent may be added once to precipitate cellulase, and then an ionic liquid and / or a poor solvent may be added so that a desired cellulase working environment is obtained. The poor solvent and the ionic liquid to be added later may be different from or the same as the original one. The cellulase working environment and its formation method and cellulose precipitation and its method are not limited to those described above, and can be implemented with appropriate modifications.

  In the cellulase working environment, as shown in FIG. 1, any cellulase may be used as long as the cellulase can exhibit the enzyme activity, and the cellulase may not necessarily be included. The cellulase working environment formation step does not necessarily require cellulose to be decomposed with cellulase. Therefore, cellulase is supplied to the cellulase working environment in a separate step, and the cellulase working environment formation step can be a preparation step for cellulose degradation by the cellulase. In carrying out the method for producing a cellulose degradation product, which will be described later, in the cellulase working environment formation step, cellulase can be supplied simultaneously with forming the cellulase working environment by adding a poor solvent containing cellulase to the ionic liquid. .

  According to the cellulose treatment method of the present invention described above, a preferable cellulase working environment for decomposing cellulose solubilized with an ionic liquid with cellulase can be easily obtained. That is, it can be decomposed by cellulase while maintaining the structural relaxation merit of cellulose solubilized with ionic liquid. In addition, the cellulase working environment can be easily formed in a system in which cellulose is solubilized with an ionic liquid. Therefore, as before, it is possible to avoid the troublesome operation of precipitating cellulose once solubilized from the ionic liquid, separating it and supplying it separately to the cellulase reaction system, and relaxing the structure of the cellulose solubilized with the ionic liquid. The inconvenience that the merit cannot be fully utilized can also be avoided. Furthermore, according to the cellulose treatment method of the present invention, the amount of cellulase used can be reduced as compared with the conventional method (Non-patent Document 1). From the above, according to the present invention, it is possible to provide a practical method for using cellulose that can efficiently decompose cellulose using an ionic liquid.

  Furthermore, according to the present invention, a preferable cellulase working environment can be obtained regardless of the characteristics of the ionic liquid (particularly, the liquidity (pH) when mixed with an aqueous poor solvent). Mixing with an aqueous anti-solvent ensures sufficient cellulase enzyme activity and corrects the pH shift that may occur when the ionic liquid and anti-solvent come into contact, or provides sufficient buffer capacity to buffer pH fluctuations. By using the poor solvent, it is possible to suppress or avoid the influence of fluctuations in the properties of the ionic liquid due to variations in impurities and manufacturing methods, and to stably form a good cellulase working environment.

(Production method of cellulose degradation products)
As shown in FIG. 2, the method for producing a cellulose degradation product of the present invention comprises the steps of preparing an ionic liquid and a cellulase working environment in the cellulose pretreatment method of the present invention, and cellulase in a cellulase working environment containing cellulase. And a step of disassembling. According to the method for producing a cellulose degradation product of the present invention, cellulose solubilized with an ionic liquid can be efficiently degraded based on what has been described in the cellulose treatment method described above. In addition, as a decomposition product of cellulose, any cellulose having a low molecular weight may be used. More specifically, there are cellobiose and cellooligosaccharide in addition to glucose which is the final degradation product.

(Cellulose decomposition process)
In order to form a cellulase working environment containing cellulase, as shown in FIG. 2, the cellulase working environment may be formed using a poor solvent containing cellulase in advance, or cellulase may be separately supplied to the cellulase working environment. May be. One or more types of cellulase may be used, or two or more types may be combined so that cellulose can be efficiently decomposed. The cellulase may be in a form held on a suitable carrier.

  Cellulose is reduced in molecular weight by decomposing cellulose with cellulase under the cellulase working environment. For this reason, although it depends on the kind of ionic liquid and poor solvent in the cellulase working environment, it gradually dissolves in the medium in which the ionic liquid and poor solvent in the cellulase working environment coexist from the deposited state. In this case, the reaction medium in the cellulase working environment is gradually clarified.

  In the method for producing a degradation product of cellulose in the present invention, the cellulose to be decomposed preferably contains crystalline cellulose. This is because the structure of crystalline cellulose is effectively relaxed by the ionic liquid.

  Each step in the method for producing a cellulose degradation product of the present invention can be carried out not only as a cellulose degradation product as a final product but also as a cellulose saccharification method. That is, according to this invention, the saccharification method of a cellulose provided with the preparation process of the ionic liquid in which the cellulose was solubilized, a cellulase working environment formation process, and a cellulose decomposition process is also provided. The cellulose degradation product obtained in the cellulose degradation step can be used as a modified body in the next step or as a carbon source. The saccharification of cellulose is not limited to decomposition into glucose, which is a constituent monosaccharide, but may be any one that can be decomposed into a low molecular weight form of cellulose, and includes those that decompose into cellobiose and cellooligosaccharide.

  The enzyme reaction conditions for decomposing cellulose with cellulase are not particularly limited. In general, it is set in consideration of the optimum pH, optimum temperature, etc. of the cellulase to be used, but the reaction temperature is 30 ° C. or more and 70 ° C. or less, and can be about 1 hour or more and 24 hours or less. Moreover, pH can be made into 2 or more and 6 or less.

  As described above, according to the method for producing a cellulose degradation product and the method for saccharifying cellulose according to the present invention, cellulose can be effectively decomposed with cellulase. It becomes available.

(Enzyme reaction medium)
The enzyme reaction medium of the present invention is a poor solvent in the relationship between an ionic liquid capable of solubilizing a substrate and the ionic liquid with respect to the substrate, and has an enzyme working environment in which the enzyme can exert its enzymatic activity in the presence of the ionic liquid. An amount of solvent that can be formed. According to such an enzyme reaction medium, the substrate solubilized in the ionic liquid retains at least a part of the solubilized state. For this reason, it is difficult to dissolve in an environment where the enzyme is activated, for example, it is complexed by the action of hydrogen bonds or van der Waals forces, or it is complexed with other substances. Even if it takes a substrate form, it can be made easy to use by acting an enzyme. Therefore, according to the enzyme reaction medium of the present invention, it can be used as an enzyme substrate regardless of the substrate form.

  The substrate is not particularly limited, and examples thereof include natural polysaccharides and natural resins, and polymers containing synthetic polysaccharides and synthetic resins. Examples of natural polysaccharides include cellulose, xylan, chitin, and chitosan. In addition, the enzyme depends on the type of substrate, but when cellulose is used as the substrate, it is the cellulase already described. Examples of xylan include xylanase, and examples of chitin and chitosan include chitinase.

  The ionic liquid is not particularly limited, and is determined according to the type of substrate, the ease of forming an enzyme action environment, and the like. It can be appropriately selected from known ionic liquids. For example, when cellulose is used as a substrate, the ionic liquid described above can be preferably used. Moreover, the ionic liquid which can be preferably used for cellulose can also be preferably used for chitin and chitosan having a similar structure.

  The enzyme working environment is sufficient if the enzyme can act on the substrate. Whether the environment in which the ionic liquid and the poor solvent coexist is an environment in which the desired enzyme for the desired substrate can operate can be determined in various forms (ionic liquid and poor solvent of the ionic liquid and poor solvent). It can be confirmed by constructing a coexistence system by type and mixing ratio) and evaluating the enzyme activity. Cellulase has already been described, but if there is a system that can measure the enzyme activity of a desired enzyme, the enzyme working environment of the present invention can be easily confirmed.

  As already explained, in order to prepare a reaction medium capable of suppressing or avoiding the adverse effects on the enzyme activity and degradation efficiency of pH shift and pH fluctuation due to fluctuations in the characteristics of the ionic liquid, the ionic liquid used and the poor solvent were in contact with each other. It is preferred to use an anti-solvent that has sufficient adjustability to correct pH shifts that may sometimes occur or buffer pH fluctuations.

  The enzyme reaction medium of the present invention may contain a substrate at least once solubilized with an ionic liquid. Such a substrate may be added to the enzyme reaction medium of the present invention in a state solubilized in the ionic liquid, or may be previously solubilized and contained in the ionic liquid at the time of forming the reaction medium. At least a part of the substrate contained in the enzyme reaction medium may be deposited. Even if the substrate is deposited, if the ionic liquid is present in the enzyme reaction medium, and once solubilized by the ionic liquid, at least the relaxed structure when solubilized by the ionic liquid is used. Retaining part makes it easier for the enzyme to approach the substrate. The enzyme reaction medium may further contain an enzyme. The enzyme may be added in advance to a poor solvent, or may be added after forming the enzyme working environment.

(Cellulose decomposition medium)
The cellulolytic medium of the present invention is a cellulase that is a poor solvent in relation to the ionic liquid that can solubilize cellulose and the ionic liquid, and in which the cellulase can exhibit enzyme activity in the presence of the ionic liquid. An amount of solvent sufficient to form a working environment. The cellulose decomposition medium of the present invention is one form of the enzyme reaction medium of the present invention.

  Regarding the cellulose, cellulase, ionic liquid and poor solvent in the cellulose decomposition medium of the present invention, various aspects already described in the cellulose treatment method and the degradation product production method of the present invention can be employed. The cellulose-decomposing medium can also contain cellulose that has been solubilized at least once by the ionic liquid, at least a portion of the cellulose can be precipitated, and can also contain cellulase.

  According to the cellulose decomposition medium of the present invention, cellulose and cellulase can be present in a state where cellulose can be efficiently decomposed by cellulase. For this reason, cellulose is useful for practical use as various carbon sources and raw materials.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to a following example.

In this example, 1-ethyl-3-methylimidazolium diethyl phosphate (manufactured by Solvent Innovation) was used as the ionic liquid, and a 10 mM citrate buffer (pH 5. 0) was mixed in various ways (volume ratio) to prepare cellulase decomposition media, and the influence of the amount of ionic liquid on cellulase activity was examined. The following table shows the mixing ratio, ionic liquid content, cellulose content, cellulase content and substrate concentration in the media of Samples 1-5. In addition, Avicel PH-101 which can be obtained as crystalline cellulose was used as cellulose, and cellulase derived from Trichoderma reesei (all of which are manufactured by Sigma-Aldrich) was used as the cellulase. The specific gravity of the ionic liquid is about 1.

  In addition, in order to match | combine the final cellulose density | concentration in each medium, the cellulose was added to the ionic liquid of the quantity shown in Table 1 in the ratio of 1-5 mass%, and it was made to melt | dissolve at 50 degreeC. Then, when the citrate buffer solution shown in Table 1 was added, film-like cellulose (regenerated cellulose) immediately precipitated in all reaction media. The precipitated cellulose film was broken by vortexing, and then 2 mg of cellulase was added to the reaction medium, and an enzyme reaction was performed at 40 ° C. The conversion rate to sugars (glucose and cellobiose) after the reaction was converted to ABEE and measured by HPLC (Yasuno et al., Biosci. Biotech. Biochem. 61, 1994-1946).

  The measurement result of the conversion rate (cellulase activity) of cellulose is shown in FIG. FIG. 3 (a) shows the conversion rate to glucose (white bar: 1 hour and black bar: 24 hours), and FIG. 3 (b) shows the conversion rate to cellobiose (white bar: 1 hour and black bar: 24 hours). ). Moreover, the time-dependent change of the cellulase activity about the sample 2 (mixing ratio 1/4 of an ionic liquid / aqueous medium) is shown in FIG.

  As shown in FIG. 3, it has been found that the conversion rate varies depending on the mixing ratio of the ionic liquid and the aqueous medium. It was also found that there is a preferable mixing ratio. When the mixing ratio of the ionic liquid / aqueous medium was 1/4, saccharification proceeded best, and the conversion rate to glucose after 24 hours was 50%. Further, at the mixing ratio, the conversion rate to cellobiose after 24 hours was 20%.

  On the other hand, it was found that when the mixing ratio of the ionic liquid and the aqueous medium is 2/3 and the ratio of the ionic liquid is higher than that, the conversion rate to glucose and the conversion rate to cellobiose are lowered.

  In the following operations, the same reagents as in Example 1 were used unless otherwise specified. In this example, the time-dependent change in cellulase activity was measured for the medium 1 (ionic liquid / aqueous medium = 1/4) obtained in Example 1. In the measurement, 800 μl of cellulase solution (10 mg / ml, 10 mM citrate buffer pH 5.0) was added to 200 μl of ionic liquid solubilized cellulose prepared in the same manner as in medium 1 of Example 1. The enzyme reaction was started. As a control, the cellulase solution was heated at 100 ° C. for 10 minutes to prepare a heat-denatured cellulase. Again, 800 μl of the cellulase was added to the medium 1, and the change in cellulase activity over time was measured. The cellulase activity was measured in the same manner as in Example 1. The results are shown in FIG.

  As shown in FIG. 4, cellobiose was mainly produced at the beginning of the reaction and then decomposed to increase the amount of glucose produced. This revealed that the cellulase retained activity for at least 24 hours in the presence of the ionic liquid. In addition, since heat-denatured cellulase showed almost no activity, the degradation and saccharification of cellulose was certainly due to the enzymatic activity of cellulase, and other cellulases also degraded cellulose in the presence of ionic liquids. It was found that it can be applied to.

  In the following operations, the same reagents as in Example 1 were used unless otherwise specified. In this example, a cellulose stock solution was prepared by dissolving cellulose at 8% by mass in an ionic liquid at 50 ° C. Furthermore, cellulase and a citrate buffer solution were added to this ionic liquid so as to have the composition shown in Table 2, and the mixture was shaken at a reaction temperature of 40 ° C. for 20 hours to evaluate cellulase activity. The cellulase activity was evaluated in the same manner as in Example 1. The results are shown in Table 2 and FIG.

  As shown in FIG. 5, it was found that the cellulase activity (conversion rate) was improved accordingly when the cellulase concentration was increased. Therefore, it was found that cellulase works sufficiently even in the presence of an ionic liquid. It was also found that the reaction can be decomposed to about 80% after 20 hours of reaction.

  In this example, an attempt was made to saccharify cellulose with cellulase at a lower concentration than in Example 3. That is, cellulose was decomposed at cellulase concentrations of 0.2 mg / ml, 0.04 mg / ml and 0.02 mg / ml. Cellulase activity was evaluated in the same manner as in Example 3 except that a cellulose stock solution was prepared at 5% by mass. The results are shown in FIGS.

  As shown in FIGS. 6 and 7, it was found that the cellulase concentration could be degraded to about 75% even at 0.2 mg / ml. Accordingly, it was found that cellulase can decompose cellulose even in a considerably low concentration in the presence of an ionic liquid. This supported that cellulase was stable even in the presence of ionic liquid, and that the rigid structure of cellulose was effectively maintained in an environment where cellulase was effectively disrupted by ionic liquid. is doing.

  In this example, acetic acid was used as the anion constituting the ionic liquid, cellulase and citrate buffer were added so as to have the composition shown in Table 3, and the treatment was performed as shown in Table 3. The cellulase activity was evaluated. The results are shown in Table 3.

As shown in Table 3, it was found that about 40% of the anion can be decomposed even if the anion is an acetate ion. That is, it was found that an environment in which cellulase can act can be formed in the same reaction field where cellulose is solubilized even with an ionic liquid having acetate ions as anions.

  In this example, the pH control effect in saccharification of cellulose was evaluated using 1-ethyl-3-methylimidazolium acetic acid as the ionic liquid. That is, Avicel PH-101 (manufactured by Flula), which is crystalline cellulose, was dissolved in the ionic liquid at 50 ° C. to a concentration of 5 wt%. As shown in Table 4, with respect to 200 mg of this solution, various concentrations of the reagent (citric acid monohydrate) (600 mM, 400 mM, 200 mM) in various aqueous solutions and 10 mM citrate buffer (pH 5. 0) 800 μl of each was added to precipitate the cellulose solubilized by the ionic liquid. The pH after reaction of each sample thus obtained is also shown in Table 4.

Next, the precipitated cellulose was crushed with vortex (trade name), and an enzyme reaction was performed using 0.2 mg of cellulase derived from T. reesei (manufactured by SIGMA). Specifically, 50 μl of a cellulase solution prepared by dissolving in 10 mM citrate buffer (pH 5.0) so as to be 4.0 mg / ml cellulase is added to the precipitated cellulose, and the enzyme reaction is performed at 40 ° C. for 24 hours. Went. After the reaction, the reaction product was derivatized with 4-aminobenzoic acid ethyl ester, and the amount of glucose and cellobiose produced were each quantified by HPLC (manufactured by Shimadzu Corporation). The HPLC measurement conditions were as follows, and the details were in accordance with the attached instruction manual. The results are shown in FIG.
(HPLC measurement conditions)
Column: ODS column Mobile phase: 0.02% TFA: acetonitrile = 85: 15
Detection wavelength: 305 nm

  As shown in FIG. 8, when a commonly used 10 mM citrate buffer was used (sample 4), the conversion efficiency from cellulase to glucose or cellobiose was about 30%, but a citric acid aqueous solution (200 mM to 600 mM). ), The conversion efficiency increased to about 68%. From the above, when using ionic liquid for solubilization of cellulose, in order to suppress or avoid the degradation and fluctuation of cellulose degradation efficiency caused by ionic liquid, it is necessary to use a buffer solution with a normal use concentration and an optimum pH of cellulase. However, it was found effective to use a poor solvent such as an aqueous solution whose pH was shifted to the acidic side from the optimum pH by increasing the reagent concentration.

  In this example, 1-butyl-3-methylimidazolium chlorine was used as the ionic liquid to evaluate the pH control effect in saccharification of cellulose. That is, Avicel PH-101 (manufactured by Flula), which is crystalline cellulose, was dissolved in the ionic liquid at 50 ° C. to a concentration of 5 wt%. As shown in Table 5, with respect to 200 mg of this solution, various concentrations of the reagent (citric acid monohydrate) (100 mM, 50 mM, 10 mM) and 10 mM citrate buffer (pH 3. 0, pH 4.0, and pH 5.0) 800 μl were added to precipitate the solubilized cellulose. The pH after reaction of each sample thus obtained is also shown in Table 5. For each of the obtained samples, an enzyme reaction was carried out in the same manner as in Example 6 and conversion efficiency was measured. The results are shown in FIG.

  As shown in FIG. 9, when a commonly used 10 mM citrate buffer (pH 5.0) was used (sample 6), the conversion efficiency from cellulase to glucose or cellobiose was less than about 80%. By using an acid concentration of 10 mM and a buffer solution having a pH of 3.0 to 4.0, a higher conversion efficiency could be obtained. From the above, when the ionic liquid is used for solubilization of cellulose, in order to suppress or avoid the decrease or fluctuation in cellulose degradation efficiency due to the ionic liquid, a buffer solution having a normal use concentration and an optimum pH of cellulase and It has been found that it is effective to use a poor solvent such as a buffer whose buffering capacity is enhanced by setting the pH to be lower than the optimum pH on the acidic side. Moreover, in the present Example, it turned out that sufficient pH adjustment cannot be performed by citric acid aqueous solution (100 mM-10 mM).

  In this example, 1-octyl-3-methylimidazolium chlorine was used as the ionic liquid to evaluate the pH control effect in saccharification of cellulose. That is, Avicel PH-101 (manufactured by Flula), which is crystalline cellulose, was dissolved in the ionic liquid at 50 ° C. to a concentration of 1 wt%. To 100 mg of this solution, as shown in Table 6, add 900 μl of a citrate buffer solution (pH 2.1) having a citrate concentration of 50 mM and a 10 mM citrate buffer solution (pH 3.0, pH 4.0 and pH 5.0). Solubilized cellulose was precipitated. The pH after reaction of each sample thus obtained is also shown in Table 6. For each of the obtained samples, the enzyme reaction was carried out in the same manner as in Example 6 except that the amount of cellulase was 2.5 mg, and the conversion efficiency was measured. Specifically, 200 μl of a cellulase solution prepared by dissolving in 10 mM citrate buffer (pH 5.0) so as to be 12.5 mg / ml cellulase was added to the precipitated cellulose, and the enzyme reaction was performed at 40 ° C. for 24 hours. Went. The results are shown in FIG.

  As shown in FIG. 10, when a commonly used 10 mM citrate buffer (pH 5.0) was used (sample 4), the conversion efficiency from cellulase to glucose or cellobiose was 17%, but the citrate concentration A higher conversion efficiency could be obtained by adjusting the pH to 10 mM and using a pH 3.0 to 4.0 buffer. On the other hand, only a low conversion efficiency was obtained with a pH 2.1 buffer. From the above, when using ionic liquid for solubilization of cellulose, in order to suppress or avoid the degradation and fluctuation of cellulose degradation efficiency caused by ionic liquid, it is necessary to use a buffer solution with a normal use concentration and an optimum pH of cellulase. However, it was found that it is effective to use a buffer solution having a buffer capacity appropriately enhanced by setting the pH to be lower than the optimum pH of cellulase within a certain range.

  In this example, the influence of imidazole impurities in cellulose saccharification using 1-ethyl-3-methylimidazolium diethylphosphate was confirmed. That is, 800 μl of 10 mM citrate buffer (pH 5.0) was added to 200 mg of the ionic liquid in which 10 mg of crystalline cellulose Avicel PH-101 (Flula) was dissolved at 50 ° C. to precipitate regenerated cellulose. At this time, imidazole was added so as to have final concentrations of 0.1, 1.0, and 5.0% (wt./vol.) In the mixed solution. As a result of measuring the pH at this time, it was pH 6.8 for imidazole 0.1%, pH 8.1 for 1.0%, and pH 8.8 for 5.0%. In the control without addition of imidazole, the pH was 5.0.

  The precipitated regenerated cellulose was crushed with a vortex, reesei-derived cellulase (manufactured by SIGMA) 0.2 mg was added, and the reaction was performed at 40 ° C. for 24 hours. Samples reacted for 1,18 and 24 hours were collected, derivatized with 4-aminobenzoic acid ethyl ester, and the amounts of glucose and cellobiose produced were quantified by HPLC (Shimadzu Corporation). As the HPLC measurement conditions, an ODS column was used, and a 0.02% TFA / acetonitrile mixture (85:15) (volume ratio) was used as the mobile phase. The details of the operation were measured at a detection wavelength of 305 nm according to the attached instruction manual.

  The influence of imidazolium salt addition on the saccharification reaction with an ionic liquid is shown in FIG. Even when the final imidazole concentration was only 0.1%, the saccharification efficiency decreased to 20% or less. In addition, when the final imidazolium concentration was 1.0% or 5.0%, the saccharification reaction did not proceed at all. From this, in the saccharification reaction in an imidazolium-based ionic liquid, the pH shifts due to the release of imidazole and the saccharification efficiency is reduced. became.

It is a figure which shows an example of the processing method of the cellulose of this invention. It is a figure which shows an example of the production method of the cellulose degradation product of this invention. It is a figure which shows the measurement result of the conversion ratio of the cellulose to the saccharide | sugar in Example 1. FIG. 3A is a diagram showing the conversion rate to glucose based on the amount of glucose in the medium, and FIG. 3B is a diagram showing the conversion rate to cellobiose based on the amount of cellobiose in the medium. is there. It is a figure which shows the time-dependent change of the conversion rate of the cellulose to the saccharide | sugar in Example 2. It is a figure which shows the relationship between the conversion rate of the cellulose to the saccharide | sugar in Example 3, and a cellulase concentration. 6 is a graph showing the conversion rate of cellulose to sugar at various cellulase concentrations in Example 4. It is a figure which shows the relationship between the conversion rate of the cellulose to the saccharide | sugar in Example 4, and a cellulase concentration. It is a figure which shows the conversion efficiency to the saccharide | sugar (glucose and cellobiose) of the cellulose in Example 6. It is a figure which shows the conversion efficiency to the saccharide | sugar (glucose and cellobiose) of the cellulose in Example 7. It is a figure which shows the conversion efficiency to the saccharide | sugar (glucose and cellobiose) of the cellulose in Example 8. It is a figure which shows the conversion efficiency to the saccharide | sugar (glucose and cellobiose) of the cellulose in Example 9.

Claims (28)

A method for treating a cellulose-containing material,
Preparing an ionic liquid in which cellulose in the cellulose-containing material is solubilized;
A cellulase in which cellulase can exhibit enzyme activity by adding a predetermined amount of a poor solvent in relation to the cellulose to the ionic liquid containing the solubilized cellulose, the ionic liquid and the poor solvent. Forming a working environment;
A processing method comprising:   The treatment method according to claim 1, wherein at least a part of cellulose is deposited in the cellulase working environment.   The processing method according to claim 2, wherein the cellulose is precipitated by adding the poor solvent to the ionic liquid containing the solubilized cellulose in the cellulase working environment forming step or prior to the step.   The processing method in any one of Claims 1-3 including controlling the pH of the said cellulase working environment.   As the poor solvent, when the ionic liquid is mixed with the poor solvent, the poor solvent has an adjustment ability to correct a shift from a preferred pH preferable for the cellulase to act, or an adjustment ability to suppress pH fluctuation with respect to the preferred pH. The processing method according to claim 4, wherein:   6. The ionic liquid according to claim 4, wherein the ionic liquid is an ionic liquid having an anion selected from a carboxylic acid anion, a halogen anion, and a phosphate anion and an imidazolium cation as constituent ions. Processing method.   The processing method according to claim 6, wherein the anion is a halogen-based anion. A method for producing a cellulose degradation product,
Preparing an ionic liquid in which cellulose in the cellulose-containing material is solubilized;
A cellulase in which cellulase can exhibit enzyme activity by adding a predetermined amount of a poor solvent in relation to the cellulose to the ionic liquid containing the solubilized cellulose, the ionic liquid and the poor solvent. Forming a working environment;
Decomposing cellulose in the cellulase working environment containing the cellulase;
A production method comprising:   The production method according to claim 9, wherein at least a part of cellulose is deposited in the cellulase working environment.   The production method according to claim 9, wherein the cellulose is precipitated by adding the poor solvent to the ionic liquid containing the solubilized cellulose in the cellulase working environment formation step or prior to the step.   The production method according to any one of claims 8 to 10, wherein the cellulase working environment contains the poor solvent in a volume ratio of 1.5 times or more with respect to the ionic liquid.   The production method according to claim 8, wherein the poor solvent is an aqueous medium.   The production method according to claim 12, wherein the poor solvent is water or a buffer solution.   The production method according to claim 8, wherein the poor solvent contains cellulase.   The production method according to claim 8, wherein the ionic liquid is non-halogen.   The production method according to claim 8, wherein the ionic liquid contains a phosphate anion or a carboxylic acid anion.   The production method according to any one of claims 8 to 16, wherein the ionic liquid contains 1-ethyl-3-methylimidazolium cation.   The production method according to any one of claims 8 to 17, wherein the cellulase includes a cellulase derived from the genus Trichoderma or Aspergillusis.   As the poor solvent, when the ionic liquid is mixed with the poor solvent, the poor solvent has an adjustment ability to correct a shift from a preferred pH preferable for the cellulase to act, or an adjustment ability to suppress pH fluctuation with respect to the preferred pH. The production method according to any one of claims 8 to 18, wherein   20. The production method according to claim 19, wherein the ionic liquid is an ionic liquid having an anion selected from a carboxylic acid anion, a halogen anion, and a phosphate anion and an imidazolium cation as constituent ions. .   The production method according to claim 20, wherein the anion is a halogen-based anion. A method for saccharification of cellulose,
Preparing an ionic liquid in which cellulose in the cellulose-containing material is solubilized;
A cellulase in which cellulase can exhibit enzyme activity by adding a predetermined amount of a poor solvent in relation to the cellulose to the ionic liquid containing the solubilized cellulose, the ionic liquid and the poor solvent. Forming a working environment;
A saccharification method comprising:   As the poor solvent, when the ionic liquid is mixed with the poor solvent, the poor solvent has an adjustment ability to correct a shift from a preferred pH preferable for the cellulase to act, or an adjustment ability to suppress pH fluctuation with respect to the preferred pH. The saccharification method according to claim 22, wherein A cellulolytic medium,
An ionic liquid capable of solubilizing cellulose;
The poor solvent in relation to the ionic liquid with respect to the cellulose, and in the presence of the ionic liquid, the poor solvent in an amount sufficient to form a cellulase working environment in which cellulase can exhibit enzyme activity;
A medium for decomposition, including   The decomposition medium according to claim 24, further comprising cellulose solubilized at least once by the ionic liquid.   26. The decomposition medium according to claim 25, wherein at least a part of the cellulose is precipitated.   The decomposition medium according to any one of claims 24 to 26, further comprising cellulase. An enzyme reaction medium,
An ionic liquid capable of solubilizing the substrate;
An anti-solvent in relation to the ionic liquid with respect to the substrate, and in the presence of the ionic liquid, an anti-solvent in an amount sufficient to form an enzyme working environment in which the enzyme can exert its enzymatic activity;
A reaction medium.