JP2012125154A - Method for producing useful substance from cellulose-containing material and use thereof - Google Patents

Abstract

The present invention provides a practical method for treating a cellulose-containing material by further decomposing cellulose using an ionic liquid.
A step of bringing a cellulose-containing material into contact with an ionic liquid to allow the ionic liquid to permeate the cellulose-containing material, a solid phase containing the cellulose-containing material, and an enzyme group containing cellulase and xylanase are brought into contact. A step of decomposing cellulose in the solid phase, and a step of producing a useful substance by fermentation of microorganisms using a carbon source containing a cellulose decomposition product obtained in the cellulose decomposition step.
[Selection] Figure 1

Description

  The present invention relates to a method for producing a useful substance from a cellulose-containing material and use thereof, and more particularly to a method for producing a useful substance from a cellulose-containing material using an ionic liquid and use thereof.

  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 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. In order to effectively use biomass as an energy source and other raw materials, it is necessary to decompose and saccharify biomass into a carbon source that can be easily used by animals and microorganisms. Problems to be solved for practical use include development of an efficient decomposition method of cellulose, which is the main component of woody or herbaceous biomass, especially crystalline cellulose.

  In the current saccharification process, biomass is pretreated by high-temperature / high-pressure treatment or acid treatment to separate cellulose, 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 pre-treatment of solubilizing cellulose with ionic liquid, cellulose after solubilization is washed with a hydrophilic solvent such as water to remove the ionic liquid, and then put into water so that it can be decomposed with cellulase Has been reported (Non-Patent Document 5).

  Furthermore, a technique has also been tried in which cellulose is swelled with an ionic liquid and then the enzyme treatment is performed after the ionic liquid is removed (Patent Documents 3 and 4, Non-Patent Document 6).

JP 2005-506401 A JP 2006-137777 A US Patent Application Publication No. US2008 / 0227162 US Patent Application Publication No. US2008 / 0190013

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 Q. Li et al., Bioresour Technol., Vol.100, p3570-3575, 2009

  Each of the above documents discloses that the ionic liquid contributes to the energy cost improvement of the pretreatment of the cellulose-containing material. However, no useful technique or material has yet been provided throughout the process of using cellulose-containing materials, in which cellulose after pretreatment with an ionic liquid is decomposed by cellulase and a useful substance is produced from the decomposition product.

  Accordingly, an object of the present disclosure is to provide a more practical method for producing a useful substance from a cellulose-containing material using an ionic liquid.

  The present inventors maintain and recover the cellulose-containing material as a solid phase by bringing the cellulose-containing material into contact with the ionic liquid so as to swell or relax the structure without completely dissolving the cellulose-containing material, It has been found that the cellulose in the solid phase can be efficiently decomposed by decomposing the cellulose in the solid phase using a specific enzyme combination. In addition, the present inventors present such a combination of enzymes on a cell surface layer of a microorganism to contact the solid phase and allow the microorganism to directly use a cellulose degradation product, thereby directly from the solid phase. The knowledge that a useful substance can be produced efficiently was obtained. The disclosure herein is provided based on these findings.

  According to the disclosure of the present specification, a method for producing a useful substance, the step of bringing a cellulose-containing material and an ionic liquid into contact with each other to cause the cellulose-containing material to permeate the ionic liquid, and a solid phase containing the cellulose-containing material And a group of enzymes containing cellulase and xylanase to decompose cellulose in the solid phase, and by fermentation of microorganisms using a carbon source containing a cellulose degradation product obtained in the cellulose degradation step A production method comprising the steps of producing a useful substance.

  The enzyme group includes one or both of Clostridium thermocellum cellobiohydrolase I (CtCel9A) and Clostridium thermocellum cellobiohydrolase I (CtCbhA), Phanerochaete chrysosporium cellobiohydrolase II (PcCBH II), Trichoderma reesei Derived endoglucanase II (TrEG II) and Trichoderma reesei-derived xylanase II (TrXyn II) may be contained. The enzyme group may further contain β-glucosidase (AaBGA) derived from Aspergillus aculeatus. The cellulose-containing material may contain crystalline cellulose.

  Prior to the decomposition step, a step of solid-liquid separation of the solid phase and the ionic liquid may be provided. In addition, at least a part of the enzymes of the group of enzymes may be supplied by the one or more microorganisms being presented on the cell surface or secreted extracellularly. In addition, any of the enzymes in the enzyme group may be supplied by the microorganisms presenting on the cell surface. Further, the ionic liquid may include 1-butyl-3-methylimidazolium acetate. The microorganism may be a yeast.

  The production method may further include a step of supplying the ionic liquid separated in the solid-liquid separation step to the permeation step.

  According to the disclosure of the present specification, an enzyme preparation for degrading a cellulose-containing material, which is one of cellobiohydrolase I (CtCel9A) derived from Clostridium thermocellum and cellobiohydrolase I (CtCbhA) derived from Clostridium thermocellum Or both contain cellobiohydrolase II from Phanerochaete chrysosporium (PcCBH II), endoglucanase II from Trichoderma reesei (TrEG II), β-glucosidase from Aspergillus aculeatus (AaBGA) and xylanase II from Trichoderma reesei (TrXyn II) An enzyme preparation is provided. The enzyme preparation preferably contains Clostridium thermocellum-derived cellobiohydrolase I (CtCbhA), and preferably contains Aspergillus aculeatus-derived β-glucosidase (AaBGA).

  According to the disclosure of the present specification, a microorganism preparation for degrading a cellulose-containing material, which is one of cellobiohydrolase I (CtCel9A) derived from Clostridium thermocellum and cellobiohydrolase I (CtCbhA) derived from Clostridium thermocellum Or both include cellobiohydrolase II (PcCBH II) from Phanerochaete chrysosporium, endoglucanase II (TrEG II) from Trichoderma reesei, β-glucosidase (AaBGA) from Aspergillus aculeatus and xylanase II from Trichoderma reesei (TrXyn II) Provided is a microbial preparation comprising one or more microorganisms that present one or more of these enzymes on the cell surface or secrete them extracellularly. The microorganism may be a yeast.

  According to the disclosure of the present specification, there is provided a method for producing a cellulose degradation product, the step of bringing a cellulose-containing material and an ionic liquid into contact with each other and allowing the cellulose-containing material to permeate the ionic liquid; Contacting the phase with an enzyme group comprising cellulase and xylanase to degrade cellulose in the solid phase.

  The ionic liquid may include 1-butyl-3-methylimidazolium acetate.

It is a figure which shows the example of the flow of the production method of the useful substance of an indication of this specification. It is a figure which shows the kind of gene and vector which are introduce | transduced into the transformed yeast produced in Example 1. FIG. It is a figure which shows the saccharification test result by the combination of a saccharification enzyme secretion type yeast culture supernatant. It is a figure which shows the result of having evaluated the relationship between the difference of a pre-processing, and the combination of an enzyme by the saccharification rate. It is a figure which shows the saccharification test result by the combination of a saccharification enzyme secretion type yeast culture supernatant. It is a figure which shows the ethanol conversion efficiency by simultaneous saccharification fermentation by the combination of a suitable saccharifying enzyme.

  The disclosure of the present specification relates to a method for producing a useful substance from a cellulose-containing material, an enzyme preparation for degrading cellulose, a microbial preparation, a method for producing a cellulose degradation product, and the like. According to the disclosure of the present specification, it is considered that at least part of the matrix containing cellulose of the cellulose-containing material is collapsed by bringing the cellulose-containing material into contact with the ionic liquid and allowing the cellulose-containing material to permeate the ionic liquid. It is done. As a result, a state is formed in which cellulase can easily access cellulose or the like as its substrate. Furthermore, after that, the solid phase containing the cellulose-containing material in such a state is recovered, and the cellulose in the solid phase can be effectively decomposed by bringing the solid phase into contact with a specific combination of enzymes. In particular, the xylanase contained in the enzyme group can decompose xylose, which is a kind of hemicellulose, which is a constituent component of the cellulose-containing matrix that is at least partially disintegrated by the ionic liquid. For this reason, disintegration of the partially disintegrated matrix is promoted, accessibility of cellulase and the like to cellulose is improved, and decomposition of cellulose is promoted. As a result, the cellulose in the solid phase can be effectively decomposed even with a small amount of enzyme produced by microorganisms.

  In addition, by supplying a solvent that is a poor solvent for the cellulose-containing material and a parent solvent for the cellulose degradation product, such as an aqueous medium, the cellulose-containing matrix infiltrated with the ionic liquid is deactivated. Cellulase can work without it. Similarly, fermentation and growth suppression of microorganisms that secrete or display such enzymes are prevented.

  Further, the ionic liquid can be reused easily and at low cost by impregnating the ionic liquid to at least partially collapse the cellulose-containing matrix and collecting the cellulose-containing material as a solid phase. Moreover, since the contact process with an ionic liquid does not require high temperature and pressure, it is a low energy cost compared with the past.

  Hereinafter, various embodiments disclosed in this specification will be described in detail with reference to the drawings as appropriate. FIG. 1 is a diagram illustrating an example of a flow of a method for producing a useful substance from a cellulose-containing material disclosed in the present specification.

(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.

  In the present specification, the cellulose-containing material may be any material that contains cellulose as described above. Accordingly, the cellulose may be crystalline cellulose or non-crystalline cellulose. The cellulose-containing material may contain so-called lignocellulose containing hemicellulose or lignin in addition to cellulose. Lignocellulose usually contains crystalline cellulose. 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 and wheat straw, agricultural waste such as rice husks, bagasse and wood chips, and waste paper And biomass (woody and herbaceous) containing various wastes such as building waste. Among these, the treatment with the ionic liquid [Bmim] [OAc] is effective for hard biomass (woody biomass).

  The cellulose-containing material applied to the various embodiments disclosed in the present specification is not particularly limited. As disclosed in the examples described later, the ionic liquid penetrates into the cellulose-containing material even if the cellulose-containing material contains crystalline cellulose or the cellulose constitutes a matrix with lignin or hemicellulose. It is known that at least the part can be collapsed. That is, like crystalline cellulose, even if it strongly interacts by hydrogen bonding to form a region with high crystallinity, the ionic liquid permeates, relaxes the structure, and promotes decomposition by cellulase. Further, according to the study by the present inventors, in the case of carrying out the permeation step with an ionic liquid, considering the action of the ionic liquid on the matrix containing cellulose, the cellulose-containing material is insoluble or hardly soluble in water. Preferably there is. Examples of the cellulose-containing material include a cellulose-containing material containing crystalline cellulose, and a material containing a cellulose-containing matrix derived from a plant cell wall containing lignin and / or hemicellulose in addition to cellulose. Typically, herbaceous or woody biomass is mentioned. The cellulose-containing material may be refined by pulverization, chopping or the like prior to the infiltration step. The cellulose-containing material preferably takes the form of a powder, but its average particle size is preferably 4 mm or less. Typically, it is preferably 1000 μm or less. The average particle size is preferably 100 μm or more in consideration of the pulverization operation, preferably 150 μm or more, and may be about 250 μm.

(Method for producing useful substances from cellulose-containing materials)
(Penetration process)
A method for producing a useful substance from a cellulose-containing material disclosed in the present specification (hereinafter, simply referred to as this production method) is a method in which a cellulose-containing material and an ionic liquid are brought into contact with each other to bring the ionic liquid into the cellulose-containing material. Infiltrating. Hereinafter, this process is referred to as an infiltration process. In this permeation step, an ionic liquid that is a liquid phase is brought into contact with a cellulose-containing material that is a solid phase. The ionic liquid is a salt that includes a cation component and an anion component and exists as a liquid even at room temperature, typically 100 ° C. or lower. The ionic liquid will be described in detail later.

  The ionic liquid is permeable to the matrix containing cellulose of the cellulose-containing material, and can disintegrate at least a part of the cellulose-containing material and relax its structure. In this specification, osmosis | permeation intends the structural relaxation of the matrix containing the cellulose in a cellulose containing material, and does not intend to melt | dissolve all the cellulose in a cellulose containing material. Therefore, it does not exclude that a part of cellulose is dissolved as a result of permeation. Although not limiting the disclosure herein, cellulose is often a polymeric material having both a hydrophobic region and a hydrophilic region. An ionic liquid contains a cation component and an anion component, and exhibits high solubility and permeability to many materials. Therefore, it is unclear which part of cellulose the ionic liquid acts on, but it has affinity for any region of cellulose, so it penetrates into the cellulosic matrix and relaxes or disintegrates at least part of it. it is conceivable that. In addition, when the cellulose-containing material has a cellulose-containing matrix containing hemicellulose or lignin in addition to cellulose, similarly, it interacts with any region of these components and penetrates into the cellulose-containing matrix, It is thought that a part of it will ease or collapse.

  The ionic liquid used in the permeation process is not particularly limited as long as it permeates the cellulose-containing material. If it is such an ionic liquid, it is thought that the matrix containing cellulose can be at least partially disintegrated by the combination of the cation component and the anion component. As such an ionic liquid, it is preferable to use a hydrophilic ionic liquid. In the present specification, the hydrophilic ionic liquid refers to an ionic liquid that is mixed with water without two-phase separation. Moreover, the hydrophilic ionic liquid should just be an ionic liquid mixed with water at least in the range of the fermentation temperature by microorganisms. Examples of the hydrophilic ionic liquid having permeability to the cellulose-containing material include an ionic liquid capable of suspending or dispersing the cellulose-containing material when mixed with the cellulose-containing material.

  The hydrophilic ionic liquid is not particularly limited, and examples thereof include quaternary ammonium salts, quaternary phosphonium salts, substituted imidazolium salts substituted with hydrocarbon groups, substituted pyridinium salts, substituted piperidinium salts (alicyclic). Quaternary ammonium salts), tertiary sulfonium salts, and the like. The ionic liquid of the present invention includes quaternary ammonium salts, quaternary phosphonium salts, substituted imidazolium salts, substituted pyridinium salts and substituted piperidinium salts. Preferably used. Quaternary ammonium salts, quaternary phosphonium salts, substituted imidazolium salts, substituted pyridinium salts and substituted piperidinium salts are represented by the general formulas (I) to (V), respectively.

  As the constituent cation species of the hydrophilic ionic liquid, as shown in the general formula (I), an ammonium cation in which four identical or different substituents are bonded to a nitrogen atom, as shown in the general formula (II), the same or A phosphonium cation in which four different substituents are bonded to a phosphorus atom, as shown in the general formula (III), an imidazolium cation in which two nitrogen atoms of the imidazole ring are bonded to the same or different substituents, a general formula (IV ) A pyridinium cation in which the nitrogen atom on the pyridine ring is bonded to a substituent, as shown in the general formula (V), a piperidinium cation in which the nitrogen atom on the piperidine ring is bonded to a substituent, the same or Examples include sulfonium cations in which three different substituents are bonded to a sulfur atom. In the cationic species having a cyclic structure, a substituent such as lower alkyl may be bonded to carbon atoms on various rings.

  Preferred constituent cation species include an imidazolium cation in which two nitrogen atoms of the imidazole ring are bonded to the same or different substituents, a pyridinium cation in which a nitrogen atom on the pyridine ring is bonded to a substituent, and a nitrogen atom on the piperidine ring. Examples include piperidinium cations bonded to a substituent. In addition, these cyclic bodies may form a polycyclic structure in which two are connected.

  The substituents in these cationic species (represented by R1 to R4 in the general formulas (I) to (V)) are each independently about 1 to 8 carbon atoms and are linear or branched And preferably an alkyl group, an alkenyl group, an alkynyl group or the like. Preferable alkyl groups include linear alkyl groups having 1 to 6 carbon atoms, more preferably 1 to 4 carbon atoms. Specific examples include a methyl group, an ethyl group, an n-propyl group, an n-butyl group, an n-pentyl group, and an n-hexyl group. In addition, the substituent may have an alkoxy group having an alkyl group having about 1 to 4 carbon atoms such as a methyl group, an ethyl group, an n-propyl group, and an n-butyl group. Preferably, an alkoxy group is provided at the end of the alkyl chain, such as a 2-methoxyethyl group.

  Examples of preferable cationic species include N-methylimidazolium, N-ethylimidazolium, 1,3-dimethylimidazolium, 1,3-diethylimidazolium, 1-ethyl-3-methylimidazolium, and 1-propyl-3. -Methylimidazolium, 1-butyl-3-methylimidazolium, 1-hexyl-3-methylimidazolium, 1,2,3-trimethylimidazolium, 1,2,3,4-tetramethylimidazolium, etc. It is done. Further, N-propylpyridinium, N-butylpyridinium, 1,4-dimethylpyridinium, 1-butyl-4-methylpyridinium, 1-butyl-2,4-dimethylpyridinium, and the like can be given. Furthermore, trimethylammonium, ethyldimethylammonium, diethylmethylammonium, triethylammonium, tetramethylammonium, triethylmethylammonium, tetraethylammonium and the like can be mentioned.

  One type of cation may be used, or two or more types may be used in combination. Preferred is an imidazolium cation. Examples include non-control imidazolium cations such as 1-ethyl-3-methylimidazolium cation, non-control imidazolium cations such as 1- (2-methoxy) ethyl-3-methylimidazolium cation, and the like.

  Examples of the anion species include a halogen anion, a carboxylate anion, a sulfonate anion, and a phosphate anion. Examples of the halogen anion include chlorine anion, bromine anion and iodine anion. Examples of the carboxylate anion include C1-C18 monocarboxylate anion and dicarboxylate anion such as formate anion, acetate anion, fumarate ioanine, oxalate anion, lactate anion and pyruvate anion. Examples of the sulfonate anion include a sulfonate anion, a methanesulfonate anion, an octanesulfonate anion, a dodecanesulfonate anion, and an eicosanesulfonate anion. Phosphate anions include phosphate anion, methyl phosphate monoester anion, ethyl phosphate monoester anion, propyl phosphate monoester anion, butyl phosphate monoester anion, methyl phosphate diester anion, ethyl phosphate diester anion, propyl phosphate diester anion And butyl phosphate diester anion. Of these, various phosphate anions, carboxylate anions, and halogen anions are preferable.

  Examples of such ionic liquids include imidazolium carboxylate, imidazolium chloride and imidazolium dialkyl phosphate, and more preferably 1-ethyl-3-methylimidazolium dialkyl phosphate, 1-ethyl-3- Methyl imidazolium formate, 1-ethyl-3-methylimidazolium acetate, 1-ethyl-2,3-dimethylimidazolium acetate, 1-butyl-3-methylimidazolium acetate, 1-ethyl-3-methylimidazolium Fumarate, 1-ethyl-3-methylimidazolium lactate, 1-butyl-3-methylimidazolium chloride, 1-ethyl-3-methylimidazolium chloride. Particularly preferably, 1-ethyl-3-methylimidazolium dialkyl (especially diethyl) phosphate, 1-ethyl-3-methylimidazolium formate, 1-ethyl-3-methylimidazolium acetate, 1-butyl-3-methyl Examples include imidazolium acetate, 1-ethyl-3-methylimidazolium chloride, and 1-butyl-3-methylimidazolium chloride.

  The ionic liquid used in the permeation step preferably contains at least 1-butyl-3-methylimidazolium acetate ([Bmim] [OAc]). [Bmim] [OAc] can be used alone, but can also be used in combination with two or more other ionic liquids as long as the action of [Bmim] [OAc] is not inhibited. According to the action of the anion and cation of [Bmim] [OAc], the crystal structure of cellulose can be effectively destroyed regardless of whether it is soft biomass or hard biomass. Further, according to [Bmim] [OAc] [, by separating [Bmim] [OAc] from the cellulose-containing material by solid-liquid separation, even if [Bmim] [OAc] remains, the cellulase does not dissolve the cellulose. It can be decomposed effectively. For this reason, the washing | cleaning process of an ionic liquid can be abbreviate | omitted or avoided, and the process process of a cellulose containing material and a decomposition | disassembly process can be simplified. [Bmim] [OAc], which is a hydrophilic ionic liquid, is a poor solvent for the cellulose-containing material and is freely mixed with a solvent that is a parent solvent for the cellulose degradation product, for example, water. For this reason, it is thought that [Bmim] [OAc] that has penetrated into the cellulose-containing material is mixed with water containing cellulase. The present inventors thought that the hydrophilic ionic liquid inhibits the enzyme reaction, and the enzyme reaction does not proceed when [Bmim] [OAc] remains in the cellulose-containing material. However, contrary to our expectation, even when [Bmim] [OAc] penetrates into the cellulose-containing material and is present in close proximity to cellulose, which is the cellulase substrate, cellulase degradation reaction of cellulase is inhibited. It was found that cellulose was effectively decomposed without being carried out.

  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 hydrophilic solvent, and as a result, may greatly affect the catalytic activity of the enzyme. According to the present inventors, for example, in the case of an ionic liquid using an imidazolium cation, the lower the purity, the easier the pH when mixed with a hydrophilic solvent such as water or a buffer solution is shifted to alkali, and cellulose by cellulase It has been found that the degradation activity tends to decrease.

  The method for bringing the cellulose-containing material into contact with the ionic liquid is not particularly limited. The cellulose-containing material only needs to be infiltrated with the ionic liquid, and the cellulose-containing material does not necessarily need to be immersed, dispersed, or suspended in the ionic liquid. In order to permeate the ionic liquid into the cellulose-containing material, for example, the cellulose-containing material may be supplied and immersed in a liquid phase of a sufficient amount of the ionic liquid, or the ionic liquid is sprayed on the cellulose-containing material. May be supplied in an amount necessary to permeate the cellulose-containing material.

  Treatment conditions for allowing the ionic liquid to penetrate into the cellulose-containing material can be set as appropriate. For example, the heating may be performed in a range of about 150 ° C. or less. Preferably it heats to 40 degreeC or more, More preferably, it is 50 degreeC or more, More preferably, it heats to 80 degreeC or more. By heating, penetration of the ionic liquid into the cellulose-containing material can be promoted. If it exceeds 150 ° C., an undesirable reaction may occur depending on the type of the cellulose-containing material, and if it is less than 40 ° C., it is difficult to obtain the effect of heating. The heating time is the size of the cellulose-containing material to be used (the average particle diameter in the case of pulverization, the average length in the case of chips, etc.) and the origin (soft biomass, hard biomass, and further Although it determines suitably according to the (including classification | category is included), it is preferable to set it as the range which does not increase melt | dissolution on the ionic liquid side of a cellulose. This is because when the dissolution of cellulose proceeds and the amount of cellulose dissolved on the ionic liquid side increases, the cellulose used in the subsequent decomposition step decreases and the utilization rate decreases. For example, the temperature is preferably 80 ° C. or higher. Preferably it is 130 degrees C or less. Although depending on the heating temperature, the heating time can be 2 hours or less. Furthermore, the heating time may be 1 hour or less, and even if it is about 30 minutes, saccharification efficiency equivalent to the treatment by 1 hour can be obtained.

  In order to promote the penetration of the ionic liquid into the cellulose-containing material, the cellulose-containing material and the ionic liquid may be mixed (stirred), pressed, pulverized, Sonication may be performed. These various treatments including heating may be employed alone or in appropriate combination. The type of treatment is appropriately changed depending on the cellulose-containing material used and the amount of ionic liquid used.

  In order to reduce the amount of ionic liquid used as much as possible, about the amount of ionic liquid necessary for permeation may be supplied to the cellulose-containing material, and as will be described later, the cellulose-containing material immersed in a sufficient amount of ionic liquid. May then be separated from the ionic liquid by solid-liquid separation means such as filtration or centrifugation.

  By carrying out such an infiltration step, the ionic liquid is infiltrated into the cellulose-containing material, and the ionic liquid is retained in the cellulose or the matrix containing cellulose, and as a result, the cellulose-containing matrix becomes loose. Conceivable. It is thought that the structure of the cellulose-containing material is relaxed by the ionic liquid, so that the hydrophilic region of cellulose is easily exposed to a solvent containing cellulase when it comes into contact with a solvent such as an aqueous medium at a later stage and is decomposed by the cellulase. It is done. In addition, it is considered that the hydrophobic region of cellulose is also easily exposed to the solvent due to relaxation by the ionic liquid and is degraded by cellulase. The cellulose-containing material into which the ionic liquid has been permeated through such a permeation step can then be subjected to cellulose degradation by cellulase.

(Solid-liquid separation process)
In this production method, a step of solid-liquid separation of the solid phase containing the cellulose-containing material and the ionic liquid can be further provided after the infiltration step. By such solid-liquid separation, the ionic liquid can be effectively recovered in a form that is convenient for reuse, and by removing excess ionic liquid from the cellulose-containing material, cellulase in the subsequent cellulose degradation process The decomposition efficiency of cellulose can be improved. In the permeation step, the solid-liquid separation step is not necessarily required when only an amount capable of penetrating the ionic liquid into the cellulose-containing material or an amount close thereto is supplied.

  The solid-liquid separation method in the solid-liquid separation step is not particularly limited. Examples include filtration, pressing, centrifugation, and sedimentation. For good separation, it may be appropriately pressed during filtration. Moreover, you may grind | pulverize, grind | pulverize, etc., carrying out solid-liquid separation of the cellulose containing material which the structure eased at least partially by the ionic liquid like pressing. By performing the solid-liquid separation step, the ionic liquid is separated from the cellulose-containing material as a liquid phase, and the cellulose-containing material is separated as a solid phase. In the separated cellulose-containing material, it is considered that the ionic liquid remains in some form in the relaxed cellulose. It is considered that the ionic liquid remaining in the cellulose-containing material has an advantageous effect on the exposure of cellulose to the enzyme in the cellulase decomposition process.

  The solid phase thus separated can be effectively decomposed by cellulase because the structure of cellulose is relaxed to a state suitable for decomposition by cellulase.

(Washing process)
The production method can also include a step of washing the solid phase with an ionic liquid parent solvent, if necessary, after the solid-liquid separation step. By doing so, the ionic liquid can be effectively removed from the solid phase. According to such a washing step, since the ionic liquid can be recovered prior to the cellulose decomposition step with cellulase, it is more advantageous than the recovery of the ionic liquid from the cellulose decomposition reaction solution containing cellulase. The utilization rate can be improved. If the ionic liquid is a hydrophilic ionic liquid, the parent solvent of the ionic liquid is, for example, an aqueous liquid and a mixed liquid of water or an organic solvent compatible with water. Examples of the organic solvent include lower alcohols having about 1 to 4 carbon atoms.

  The washing step is, for example, a method of supplying the ionic liquid parent solvent used for the solid phase, bringing it into contact with the solid phase, and then recovering the ionic liquid parent solvent using the solid-liquid separation method described above as appropriate. Can be adopted.

(Cellulose decomposition process)
The cellulose decomposition step is a step of decomposing cellulose in the solid phase by bringing the separated solid phase into contact with an enzyme group containing cellulase and xylanase. Cellulase is a general term for various enzymes that act to hydrolyze cellulose to glucose. As cellulases, β1,4-endoglucanase (endoglucanase; EC 3.2.1.4), glucan 1,4-β glucosidase (EC 3.2.1.74), cellulose 1,4-β are narrowly defined. Examples thereof include cellobiosidase (cellobiohydrolase; EC 3.2.1.91), β-glucosidase (EC 3.2.1.21) and the like. 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 Clostridium genus, Trichoderma genus, Aspergillus genus, and Phanerochaete genus can be used preferably. A thermostable cellulase that exhibits high activity at 70 ° C. or higher and maintains activity at 90 ° C. to 100 ° C. can also be used. For example, it may be a cellulase derived from a hyperthermophilic archaea represented by the genus Pyrococcus. 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 cellulase may be in a form held on a suitable carrier.

(Cellobiohydrolase)
For example, cellobiohydrolase (CBH) includes CBH belonging to GHF6. CBH belonging to GHF6 is generally considered to be type II (CBH II) that produces cellobiose by cleaving cellulose from its non-reducing end. As CBH belonging to GHF6, those derived from various microorganisms are known (http://www.cazy.org/fam/GH6.html). Examples include CBH derived from P. chrysosporium, A. oryzae, and T. reesei. For example, PcCBHII consisting of the amino acid sequence represented by SEQ ID NO: 2 (Accession No .: AAB32942) derived from P. chrysosporium, and the amino acid sequence represented by SEQ ID NO: 4 derived from T. reesei (Accession NO .: AAA34210) TrCBH II consisting of

  In this specification, for example, “CBH derived from P. chrysosporium” refers to CBH produced by a microorganism classified into P. chrysosporium (which may be a wild strain or a mutant strain) or the microorganism. CBH obtained by a genetic engineering technique using a gene encoding a protein produced by Therefore, CBH, which is a recombinant protein produced by a transformant into which a gene encoding CBH (or a modified gene thereof) obtained from P. chrysosporium has been introduced, also corresponds to CBH derived from P. chrysosporium. Therefore, “CBH derived from P. chrysosporium” includes CBH that is the same genera as P. chrysosporium and is a heterogeneous strain or the same species and obtained from another strain.

  Examples of CBH include CBH belonging to GHF7 and GHF9. Such CBH is generally considered to be type I (CBH I), which generates cellobiose by cleaving cellulose from its reducing end. As CBH belonging to GHF7, those derived from various microorganisms are known (http://www.cazy.org/fam/GH7.html). Examples of CBH belonging to GHF9 include cellobiohydrolase derived from Clostridium thermocellum. Examples thereof include CtCel9K consisting of an amino acid sequence represented by SEQ ID NO: 6 (Accession No .: ABN51650) and CtCbhA consisting of an amino acid sequence represented by SEQ ID NO: 8 (Accession NO .: CAA56918).

(Endoglucanase)
Examples of endoglucanase (EG) include EG belonging to GHF5. As the first EG belonging to GHF5, those derived from various cellulase-producing microorganisms such as Trichoderma, Phanerochaete and Aspergillus are known (http://www.cazy.org/fam/GH5.html). . Especially, it can be set to 1 or 2 or more selected from EG derived from T. reesei. An example is TrEG II derived from T. reesei consisting of the amino acid sequence represented by SEQ ID NO: 10 (Accession NO .: ABA64553). Examples of EG include Cel8C, which is an endoglucanase belonging to GHF8, derived from Clostridium cellulolyticum, and consisting of the amino acid sequence represented by SEQ ID NO: 12 (Accession No .: AAA73867.1).

  Furthermore, as EG, it belongs to GHF12, is derived from T. reesei, and is represented by SEQ ID NO: 14 (EG No .: ABV71388), TrEGIII, belonging to GHF12, derived from Phanerochaete chrysosporium, SEQ ID NO: 16. PcEGIII consisting of the amino acid sequence represented (see JP 2009-247324 A), AnEGIII belonging to GHF12 and derived from Aspergillus niger and represented by SEQ ID NO: 18 (Accession No .: CAA11964).

(β-glucosidase)
The β-glucosidase (BGL) is not particularly limited, and those derived from various cellulase-producing microorganisms can be used. For example, AaBGA consisting of an amino acid sequence (Accession No .: BAA10968) derived from Aspergillus aculeatus and represented by SEQ ID NO: 20 can be mentioned.

(Xylanase)
Xylanase is an enzyme that hydrolyzes the β-1,4-bond of β-1,4-D-xylan, which is a constituent of hemicellulose, and is an enzyme included in hemicellulase. A xylanase derived from a cellulase-producing microorganism can be used. For example, xylanase II consisting of an amino acid sequence (Accession No: AAB50278) derived from T. reesei and represented by SEQ ID NO: 22 can be mentioned.

  A preferred enzyme to be used in this production method is a protein having a certain relationship with known or new sequence information such as a certain amino acid sequence specified for the enzyme and having a specific enzyme activity. May be. For example, a protein comprising an amino acid sequence in which one or several amino acids have been deleted, substituted or added in the amino acid sequence of the exemplified enzyme can be mentioned. Any one type of amino acid mutation, ie, deletion, substitution or addition, for each amino acid sequence may be used, or two or more types may be combined. The total number of these mutations is not particularly limited, but is preferably about 1 or more and 10 or less. More preferably, it is 1 or more and 5 or less. As an example of amino acid substitution, conservative substitution is preferable, and specific examples include substitution within the following groups. (Glycine, alanine) (valine, isoleucine, leucine) (aspartic acid, glutamic acid) (asparagine, glutamine) (serine, threonine) (lysine, arginine) (phenylalanine, tyrosine). Moreover, the protein which has an amino acid sequence which has 70% or more identity with respect to the specific amino acid sequence disclosed about the illustrated enzyme, and has cellulase activity is mentioned. The identity is preferably 80% or more, more preferably 85% or more, still more preferably 90% or more, and still more preferably 95% or more. Most preferably, it is 98% or more. Furthermore, it is encoded by DNA that hybridizes under stringent conditions with DNA consisting of a base sequence complementary to DNA consisting of a base sequence encoding the specific amino acid sequence disclosed for the exemplified enzyme, and has a unique cellulase activity. Examples include proteins. The stringent condition refers to, for example, a condition in which a so-called specific hybrid is formed and a non-specific hybrid is not formed. For example, nucleic acids having high base sequence identity, that is, 80% or more, preferably 85% or more, more preferably 90% or more, even more preferably 95% or more, and even more preferably 98% or more identity with a predetermined base sequence. A condition is that the complementary strands of DNA consisting of a base sequence having a hybridize and the complementary strands of nucleic acids with lower homology do not hybridize. More specifically, the sodium salt concentration is 15 to 750 mM, preferably 50 to 750 mM, more preferably 300 to 750 mM, the temperature is 25 to 70 ° C., preferably 50 to 70 ° C., more preferably 55 to 65 ° C., formamide The condition is that the concentration is 0 to 50%, preferably 20 to 50%, more preferably 35 to 45%. Furthermore, under stringent conditions, the filter washing conditions after hybridization are usually such that the sodium salt concentration is 15 to 600 mM, preferably 50 to 600 mM, more preferably 300 to 600 mM, and the temperature is 50 to 70 ° C., preferably It is 55-70 degreeC, More preferably, it is 60-65 degreeC. From the above, as yet another embodiment, the predetermined base sequence and 80% or more, preferably 85% or more, more preferably 90% or more, still more preferably 95% or more, and even more preferably 98% or more. And a protein having a unique enzyme activity derived from a parent sequence and encoded by a DNA having a nucleotide sequence having the same identity.

  In the present specification, identity or similarity is a relationship between two or more proteins or two or more polynucleotides determined by comparing sequences, as is known in the art. . In the art, “identity” means between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of such sequences. Means the degree of sequence invariance. Similarity is also the correlation between protein or polynucleotide sequences, as determined by alignment between protein or polynucleotide sequences, or in some cases by alignment between a series of partial sequences. Means the degree of More specifically, it is determined by sequence identity and conservation (substitutions that maintain specific amino acids in the sequence or physicochemical properties in the sequence). The similarity is referred to as Similarity in the BLAST sequence homology search result described later. The method for determining identity and similarity is preferably a method designed to align the longest between the sequences to be compared. Methods for determining identity and similarity are provided as programs available to the public. For example, BLAST (Basic Local Alignment Search Tool) program by Altschul et al. (Eg Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ., J. Mol. Biol., 215: p403-410 (1990), Altschyl SF, Madden TL, Schaffer AA, Zhang J, Miller W, Lipman DJ., Nucleic Acids Res. 25: p3389-3402 (1997)). The conditions for using software such as BLAST are not particularly limited, but it is preferable to use default values.

(Enzyme preparation suitable for degradation of cellulose-containing materials that have been infiltrated with ionic liquid)
These cellulases and xylanases can be used as enzyme preparations containing cellulases and xylanases. More preferably, it is an enzyme preparation containing all of cellobiohydrolase, endoglucanase and β-glucosidase, and further containing xylanase. More preferably, cellobiohydrolase I derived from Clostridium thermocellum (CtCBH I) (CBHA), cellobiohydrolase II derived from Phanerochaete chrysosporium (PcCBH II), endoglucanase II derived from Trichoderma reesei (TrEG II), β derived from Aspergillus aculeatus It is an enzyme preparation containing glucosidase (AaBGA) and xylanase II (TrXyn II) derived from Trichoderma reesei. Such an enzyme preparation is an enzyme preparation suitable for a cellulose-containing material after osmotic treatment with an ionic liquid. Furthermore, the formulation may contain one or more endoglucanase III. This enzyme preparation may be a culture supernatant obtained by culturing the microorganism preparation described below, or a roughly purified product thereof.

(Microbial preparation suitable for degradation of cellulose-containing materials that have been infiltrated with ionic liquid)
These cellulases and xylanases may be supplied by microorganisms. That is, the form secreted out of the cell by the microorganism used in the subsequent fermentation process or the form presented on the cell surface may be used. By providing cellulase and xylanase in such a form, the microorganisms can efficiently and directly use glucose or the like, which is a cellulose degradation product. Therefore, CBP (Saccharification and Fermentation Linkage Process) that simultaneously proceeds the cellulose degradation step and the fermentation step is performed. It can be realized with high efficiency and at low cost without using a commercially available enzyme preparation.

  This microorganism preparation is suitable for saccharification and fermentation of cellulose-containing materials after osmotic treatment with an ionic liquid, wherein one or more microorganisms that can secrete an enzyme group containing cellulase and xylanase outside the cell or can be presented on the cell surface It is a microbial preparation. According to this microbial preparation, this microbial preparation of this microorganism more preferably comprises one or more microorganisms that produce all of cellobiohydrolase, endoglucanase and β-glucosidase and xylanase as cellulases. More preferably, cellobiohydrolase I derived from Clostridium thermocellum (CtCBH I) (CBHA), cellobiohydrolase II derived from Phanerochaete chrysosporium (PcCBH II), endoglucanase II derived from Trichoderma reesei (TrEG II), β derived from Aspergillus aculeatus One or more microorganisms that produce xylanase II (TrXyn II) derived from glucosidase (AaBGA) and Trichoderma reesei may be included. Furthermore, it may contain one or more microorganisms that produce endoglucanase III.

  The microbial preparation preferably contains a microorganism that presents cellulase and / or xylanase on the cell surface. By using such a microorganism, a degradation product of cellulose can be efficiently used with a small amount of cellulase or the like. In addition, it is preferable to present all preferred cellulase and xylanase combinations on the cell surface of a single microorganism.

  When such a microbial preparation is used in the cellulose decomposition step, a fermentation step is also realized at the same time. Therefore, the solid phase and the microorganism preparation are brought into contact with each other in a hydrophilic solvent having a composition capable of acting, such as cellulase, and a composition suitable for the growth or proliferation of the microorganism to be used, typically a liquid that can serve as a culture medium for the microorganism to be used. Like that. In addition, about microorganisms, it demonstrates in a fermentation process.

  In order to secrete cellulase or xylanase to the outside of yeast or the like, a product obtained by adding a known secretory signal peptide to cellulase or the like may be produced in yeast. For example, a signal peptide that functions in yeast may be added to secretory production of proteins in yeast. For example, signal peptide sequences derived from yeast include, for example, yeast invertase leader, alpha factor leader, Rhizopus oryzae and C. albicans glucoamylase leader. Moreover, cellulase is hold | maintained on cell surface layers, such as yeast, with various forms. One is a form in which cellulase is retained on the yeast cell surface as it is using a known yeast cell surface display system. Two or more types of cellulase and xylanase may be held on the cell surface of the same yeast, or may be held on the cell surface of different yeasts.

  When cellulase or the like is presented on the cell surface of yeast, it is preferable that the cellulase or the like further has a cell surface binding domain necessary for cell surface presentation. As the yeast surface display system, for example, α-agglutinin which is a surface protein or a receptor thereof can be used. For example, in addition to the secretion signal, a peptide comprising 320 amino acid residues on the C-terminal side of α-agglutinin which is an aggregating protein is used. Polypeptides and methods for displaying a desired protein on the cell surface are disclosed in WO01 / 79483, JP2003-235579, WO2002 / 042483, WO2003 / 016525, and JP2006-136223. Publication, Fujita et al. (Fujita et al., 2004. Appl Environ Microbiol 70: 1207-1212 and Fujita et al., 2002. Appl Environ Microbiol 68: 5136-5141.), Murai et al., 1998. Appl Environ Microbiol 64: 4857-4861 Is disclosed. For example, the signal sequence may be an element incorporated into the vector, but may be a part of the cellulase gene. A protein cell surface display system using agglutinin can be obtained, for example, from Invitrogen as a display kit for yeast containing pYD1 vector and EBY100 Saccharomyces cerevisiae. As the cell surface display system, a system using cell surface proteins such as SAG1 and FLO1 to FLO11 can be used. Further, the method described in Japanese Patent Application Laid-Open No. 2008-263975 may be employed.

  Another form in which cellulase is retained on the surface of yeast cells is a form in which the cellulase is retained via a protein derived from the cellulosomal scaffolding protein. For cell surface display using cellulosome, methods disclosed in JP-A-2009-33993 and JP-A-2009-142260 can be employed. That is, a cellulosomal scaffolding protein-derived protein is displayed on the surface of yeast as a skeletal protein for cellulase retention. On the other hand, cellulase can be displayed on the surface via a backbone protein by adding a dockerin domain to cellulase so that it can bind to such a backbone protein.

  Cellulosome is formed outside the cells by anaerobic bacteria or anaerobic filamentous fungi, and is usually bound to the surface of the microorganism or present in the culture solution. Cellulosomes include various anaerobic microorganisms, such as cellulosomes produced by known cellulosome-producing microorganisms, cellulosomes that will be revealed in the future, and sources of scaffold proteins for retaining cellulase. Can be used as In view of the high cellulose decomposing ability and the like, cellulosomes produced by thermophilic anaerobic microorganisms such as clostridium thermocellum, clostridium bacteria such as clostridium cellulolyticum, or modified versions thereof can be used.

  In order to retain cellulase with respect to the scaffold protein for retaining cellulase, cellulase or the like can be retained in the scaffold protein by secreting and producing cellulase outside the cell with yeast or the like displaying the scaffold protein on the surface. In addition, cellulase produced outside the cell may be held in contact with the yeast in which the scaffold protein for retention is expressed on the cell surface, and retained in the scaffold protein.

  A gene encoding cellulase can be obtained based on the base sequence of cellulase obtained from a database. That is, it can be obtained as a nucleic acid fragment by PCR amplification or hybridization using DNA extracted from a predetermined yeast, nucleic acid derived from various cDNA libraries or genomic DNA libraries as a template. Alternatively, the cellulase gene may be synthesized as a nucleic acid fragment by various nucleic acid sequence synthesis methods known in the art such as chemical synthesis methods.

  General operations necessary for the production of a recombinant vector for genetic modification of microorganisms and the handling of yeast as a recombinant host are routinely performed by those skilled in the art. A person skilled in the art can carry out this method by referring appropriately to an experimental book by Maniatis, J. Sambrook et al. (Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 1982, 1989, 2001). Various operations for expressing foreign proteins by introducing such genes into various cells including yeast should follow protocols such as Molecular Cloning A Laboratory Manual second edition (Maniatis et al., Cold Spring Harbor Laboratory press. 1989). it can. In addition, as a method for introducing a vector, various conventionally known methods such as calcium phosphate method, transformation method, transfection method, conjugation method, protoplast method, electroporation method, lipofection method, lithium acetate method or other methods Is mentioned. Such a method is described in the above-mentioned experiment document. Yeast that expresses a necessary protein can be obtained by selection using a marker gene and selection by active expression for yeast or the like into which a vector has been introduced.

  The action of xylanase together with cellulase on the solid phase in the cellulose decomposition step is extremely effective for the decomposition of cellulose in the solid phase in which an ionic liquid is permeated to relax the matrix containing cellulose. According to the present inventors, an enzyme group containing cellobiohydrolase, endoglucanase and β-glucosidase is applied to a solid phase containing a cellulose-containing material infiltrated with an ionic liquid, and the amount thereof is 10 to 100 times. Despite the aim of increasing the decomposition efficiency by increasing the amount to twice, no improvement was obtained in the decomposition efficiency. On the other hand, in addition to these four types of cellulases, the addition of a very small amount (about 0.1 μg) of xylanase was confirmed to improve the decomposition efficiency, and further, the improvement of the decomposition rate according to the addition amount was confirmed. This is because the ionic liquid penetrates into the matrix containing cellulose and the structure is relaxed, but xylose maintained with cellulose in the solid phase is decomposed by xylanase, and the accessibility of cellulase to cellulose is further improved, and the decomposition efficiency Is considered to have improved.

  For the cellulose-containing material after the permeation treatment, it is preferable to carry out the decomposition step by supplying cellulase to the cellulose-containing material after the solid-liquid separation from the viewpoint of efficient recovery and reuse of the ionic liquid.

  In the cellulose-containing material after the permeation treatment, the ionic liquid is retained in the relaxed cellulose or the cellulose-containing matrix. Even after solid-liquid separation, it can be said that the ionic liquid remains retained in the cellulose or cellulose-containing matrix. Even if cellulase is decomposed into cellulose in such a state, the adverse effect of the ionic liquid on cellulase is suppressed, or the cellulase's attack improvement effect due to the relaxation of the cellulose structure by the ionic liquid is effective. It is thought that the adverse effects on Therefore, it can be said that this cellulose decomposition | disassembly process is a process which decomposes | disassembles the cellulose in a cellulose containing material with a cellulase in presence of an ionic liquid, when not implementing a washing | cleaning process.

  Cellulase is preferably contacted with the solid phase in the presence of a poor solvent for the cellulose-containing material and a parent solvent for the degradation product of cellulose. By using these solvents, cellulose degradation products can be secured in the liquid phase of the solvent, and undegraded parts of cellulose-containing materials and residues that cannot be essentially degraded by cellulase (eg, lignin) are retained in the solid phase. can do.

  As such a solvent, a hydrophilic solvent can usually be used. In addition, as the hydrophilic 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 suitable pH of general cellulase and xylanase is about 4-6, for example, citrate buffer (citric acid and sodium citrate), acetate buffer (acetic acid-sodium acetate), citrate-phosphorus Acid buffer (citric acid-sodium dihydrogen phosphate) and the like can be mentioned. It is preferable to appropriately adjust the concentration and pH of such a buffer solution and set the pH of the hydrophilic solvent phase to 4 or more and 6 or less, more surely 4.0 or more and 6.0 or less. In addition, it is preferable to use a hydrophilic solvent having a sufficient pH adjusting ability in order to suppress or avoid a pH shift caused by fluctuations in the characteristics of the hydrophobic ionic liquid or adverse effects on enzyme activity and degradation efficiency due to pH fluctuations. The hydrophilic solvent may contain an acid, an alkali or a salt for forming an appropriate salt concentration. Metal ions necessary for the enzymatic reaction can also be included.

  The temperature and time for cellulose degradation by cellulase are not particularly limited. Although it depends on the type of cellulase, it is usually about 30 ° C. to 70 ° C., preferably about 35 ° C. to 45 ° C., pH 2 or more and about 6 or less, and is performed for several hours to several tens of hours. When a thermophilic cellulase is used, it may be 70 ° C. or higher and 100 ° C. or lower.

  According to the present cellulose decomposition step, the structure of the cellulose matrix is relaxed by the ionic liquid and the cellulose and xylose are exposed at the interface with the hydrophilic solvent, so that the xylanase is likely to attack the xylose. In addition, cellulase can easily attack cellulose, and cellulase in a hydrophilic solvent can efficiently decompose cellulose. And the low molecular weight product (decomposition product containing disaccharides or oligosaccharides) of cellulose such as glucose obtained as a result of decomposition can be dissolved in a hydrophilic solvent and recovered from the hydrophilic solvent. In addition, as cellulose and xylose are reduced in molecular weight, new cellulase and xylanase access sites are exposed to the hydrophilic medium, so that degradation by cellulase is promoted. In particular, when the ionic liquid used is a hydrophilic ionic liquid, the cellulose and xylose in the cellulose-containing material are easily exposed to a hydrophilic medium in which cellulase is present. These intermediate degradation products are further reduced in molecular weight by cellulase or xylanase in a hydrophilic solvent. In addition, carbon sources other than cellulose derived from cellulose-containing materials such as hemicellulose and lignin other than xylose remain in the hydrophilic medium as a solid phase.

  From the above, by carrying out the cellulose decomposition step, a cellulase decomposition residue as a solid phase, a cellulose decomposition product and a xylose decomposition product as solutes are obtained in a hydrophilic solvent.

(Cellulose degradation product recovery process)
After carrying out the cellulose decomposition step, a cellulose decomposition product recovery step can be carried out as necessary. By recovering the hydrophilic solvent phase by an appropriate solid-liquid separation means, a decomposition product such as cellulose can be easily separated from the decomposition residue. If the recovered hydrophilic solvent phase is a cellulose-containing material that has been subjected to a decomposition step after solid-liquid separation of the ionic liquid, the concentration of the ionic liquid is sufficiently reduced. It can utilize for the fermentation process etc. in the production method of the useful substance to explain. Moreover, you may concentrate as needed. Cellulase is contained in the hydrophilic solvent phase, but cellulase can be separated and recovered by a known method, for example, by immobilizing cellulase on a solid phase.

(Fermentation process)
A fermentation process is a process of producing a useful substance by fermentation of microorganisms using a carbon source containing a decomposition product such as cellulose obtained in the cellulose decomposition process. The cellulose degradation product used in this production method may be separated and recovered from the hydrophilic solvent phase after the cellulose decomposition step, its concentrate, or the hydrophilic solvent phase. As described above, when the content of the ionic liquid is sufficiently reduced, the hydrophilic solvent phase can be used for the fermentation process even if it is as it is. Therefore, for example, the hydrophilic solvent phase after the cellulose decomposition step and the like can be used as a part of the fermentation medium. In addition, although the collection process of a non-cellulose fraction may be implemented prior to a fermentation process, the collection process of a non-cellulose fraction can also be implemented after a fermentation process.

  The microorganism used in the fermentation process is not particularly limited, and is appropriately selected according to the type of useful substance that can assimilate the degradation product of cellulose and is to be produced. The cellulose degradation product is mainly glucose, but it may be disaccharide, oligosaccharide, hemicellulose-derived xylan, etc. depending on the embodiment of the degradation process. For example, fungi such as yeast and mold, and microorganisms such as E. coli. The microorganism may be a wild type, artificially modified so as to efficiently assimilate cellulose degradation products by genetic engineering techniques, etc., or modified so that useful substances can be produced. Also good.

  For example, the yeast is not particularly limited, and various known yeasts can be used. Saccharomyces cerevisiae and other Saccharomyces yeasts, Schizosaccharomyces pombe Schizosaccharomyces pombe yeasts, Candida krusei, Candida shehatae, etc. Candida yeast, Pichia pastoris, Pichia stipitis, Pichia yeast, Hansenula yeast, Trichosporon yeast, Brettanomyces genus Yeast, yeast of the genus Pachysolen, yeast of the genus Yamadazyma, yeasts of the genus Kluyveromyces such as Kluyveromyces marxianus and Kluveromyces lactis It is done. Of these, Saccharomyces yeasts are preferable from the viewpoint of industrial availability and the like, and Saccharomyces cerevisiae is more preferable.

In addition, yeast that produces a compound that also becomes an organic raw material such as an organic acid such as lactic acid or C3 to C5 alcohol by genetic engineering modification can be used. According to such yeast, useful substances can be directly produced using cellulose as a raw material. For example, transformed yeasts such as lactic acid-producing yeast are disclosed in, for example, JP-A-2003-334092, JP-A-2004-187743, JP-A-2005-137306, JP-A-2006-6271, JP-A-2006-20602, JP-A-2006-42719, JP-A-2006-75133, JP-A-2006-296377, etc., and these transformed yeasts can be used in the present invention. All of the contents described in these publications are incorporated herein by reference.
Typically, yeast such as S. cerevisiae that produces ethanol can be mentioned. Moreover, the yeast and lactic acid bacteria which produce an organic acid may be sufficient.

  In the cellulose decomposition step, when a microbial preparation is used, the cellulose decomposition step is also a fermentation step.

  What is necessary is just to implement a fermentation process according to the kind of microorganisms to be used and the useful substance which it is going to produce. For the culture for fermentation, stationary culture, shaking culture, aeration-agitation culture, or the like can be used. The aeration conditions can be appropriately selected from anaerobic conditions, microaerobic conditions, aerobic conditions, and the like. The culture temperature is not particularly limited, but may be in the range of 25 ° C to 55 ° C. Moreover, although culture | cultivation time is also set as needed, it can be set as several hours-about 150 hours. The pH can be adjusted using an inorganic or organic acid, an alkaline solution, or the like. During the culture, antibiotics such as ampicillin and tetracycline can be added to the medium as necessary. In addition, after completion | finish of a conversion process, you may implement the process of removing microorganisms from a culture solution, collect | recovering fractions containing useful substances, such as ethanol, and also concentrating this.

  Although it does not specifically limit as a useful substance, The thing which can produce | generate microorganisms using glucose is preferable. For example, lower alcohols such as ethanol, propanol, isopropanol, butanol and isobutanol, fine chemicals (coenzyme Q10, vitamins and their raw materials, etc.) by adding an isoprenod synthesis route, organic acids such as lactic acid, glycerin and plastics by modifying glycolysis -Materials targeted by biorefinery technology, such as chemical raw materials.

(Recovery process of non-cellulose fraction)
At any timing after the cellulose decomposition step, for example, before or after the fermentation step, the solid phase residue in the hydrophilic solvent can be recovered by solid-liquid separation as necessary. The recovered solid phase is a decomposition residue by cellulase and xylanase, and is a non-cellulose fraction in the cellulose-containing material (typically lignin, and if no hemicellulase other than xylanase is used, micellulose other than hexylose is used. Is also included). Since this residue contains lignin, which is an aromatic polymer, at a high rate, and excessive condensation is suppressed, it can be used for various applications. Moreover, a phenol type compound can also be obtained by making it contact with the enzyme which decomposes | disassembles a lignin. Furthermore, when the solid phase residue contains cellulose, it can be contacted with cellulase again, and the cellulose decomposition step can be repeated to improve the utilization rate of cellulose.

(Ionic liquid supply (reuse) process)
The production method may further include a step of supplying and reusing the ionic liquid separated from the solid phase containing the cellulose-containing material to the contact step at an arbitrary timing after the solid-liquid separation step. Reusing the ionic liquid can greatly reduce the cost of the entire process. The ionic liquid separated and recovered as a liquid phase in the solid-liquid separation step can be supplied again to the infiltration step and used. In the reuse process, the ionic liquid recovered in the cleaning process can be used together. In the washing step, since the ionic liquid is contained in a state dissolved in the parent solvent, it is preferable to remove or separate the parent solvent as appropriate when reused in the permeation step.

  As described above, according to the useful substance production method disclosed in the present specification, the cellulose degradation product obtained more efficiently than the conventional method is used, so the production cost of the useful substance is effectively reduced. Can be reduced.

(Production method of degradation products of cellulose-containing materials)
According to the indication of this specification, the production method of the decomposition product of a cellulose containing material provided with the said osmosis | permeation process and the process of decomposing | disassembling the cellulose in a cellulose containing material using a cellulase is provided. According to the production method disclosed in the present specification, cellulose and xylose in a cellulose-containing material are relaxed to a state suitable for cellulase degradation by an ionic liquid, and are easily exposed to cellulase and xylanase. It is efficiently degraded by cellulase and xylanase. In addition, even if the ionic liquid remains in the cellulose-containing material, the adverse effects on the enzyme reaction are avoided or suppressed, so it is not necessary to remove the ionic liquid with high accuracy, thus simplifying the process. it can.

  The various modes described for the production method of useful substances can be applied as they are to the permeation step and the degradation step in the method for producing a cellulose degradation product disclosed in this specification. Moreover, you may implement a solid-liquid separation process, a washing | cleaning process, and a reuse process suitably. 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.

  As described above, according to the method for producing a cellulose degradation product disclosed in this specification, it is easy to recover the used ionic liquid and degradation products such as cellulose and xylose. Moreover, cellulose can be effectively decomposed with a small amount of enzyme by using xylanase. From the above, it becomes possible to use a more practical cellulose-containing material.

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

(1) Construction of saccharogenic enzyme-producing yeast (part 1)
For the purpose of examining a combination of cellulolytic enzymes suitable for ionic liquid-treated biomass, a genetically modified yeast characterized by secreting and producing cellulase was prepared. Escherichia coli JM 109 strain (Toyobo) was used for vector construction, and LB medium was used for culture. The insert fragment introduced into the vector was obtained by PCR from the genomic DNA of Saccharomyces cerevisiae YPH 499 strain (Stratagene, MATa, ura3-52, lys2-801, ade2-101, trp1-Δ63, his3Δ200, leu2Δ1).
In addition, the YPD medium (Yeast extrsct 10 g / l, Pepton 20 g / l, D-Glucose 20 g / l) was used for culture | cultivation of S. cerevisiae YPH499 strain.

  A yeast chromosome transfer vector was constructed based on pAUR112, a commercially available vector. The Saccharomyces cerevisiae TDH3 promoter, secretion signal, multiple cloning site, and CYC1 terminator were sequentially introduced into this vector, and the resulting final vector was named pAUR112-GAPSSRG vector.

Seven saccharifying enzyme genes shown in the following table were obtained by PCR. As shown in FIG. 2, the obtained PCR amplified fragment was introduced into the pAUR112-GAPSSRG vector by In Fusion PCR Cloning (Clontech). The ligation solution was transformed into E. coli (ECOS ^™ Competent E. coli DH5α; Nippon Gene) and each clone grown on an LB plate supplemented with ampicillin to a final concentration of 50 μg / ml was subjected to colony PCR. A clone was selected. The target clone was cultured in an LB culture solution containing ampicillin, plasmid DNA was extracted from the main culture solution, and this mixed DNA solution was used in the subsequent experiments. Moreover, it was confirmed by the gene sequence analyzer ABI PRISM 310 Genetic Analyzer (Applied Biosystems) whether it was the target vector.

  Host yeast was cultured with shaking in 2 ml of YPD broth at 30 ° C, and a competent cell was prepared using the Frozen-EZ Yeast Transformation II kit (ZYMO RESERCH). . Subsequently, about 0.5 μg of plasmid DNA solution was transformed according to the protocol, and the plate seeded on the selection medium was statically cultured at 30 ° C. As the selection medium, aureobaticin selection medium was used. The obtained colonies were sown again in a new selection medium, and those showing stable characteristics were selected.

  Each genetically modified yeast is shaken and cultured in 2 ml of YPD culture solution at 30 ° C for 17 hours. The resulting cells are collected, and then genomic DNA is prepared according to Gen Toru-kun for yeast (Takara Bio). did. Using the adjusted genome as a template, it was confirmed by PCR whether the target saccharifying enzyme gene was introduced. For the PCR reaction, Ex-Taq DNA polymerase (Takara Bio) was used as an amplification enzyme. The reaction composition was in accordance with the standard method. PCR reaction conditions were as follows: heat treatment at 96 ° C for 2 minutes followed by three temperature changes of 96 ° C for 30 seconds, 53 ° C for 30 seconds, and 72 ° C for 60 seconds. This was repeated 25 cycles, and the final temperature was 4 ° C. A dye was added to the reaction solution, and the presence or absence of the target fragment was confirmed by electrophoresis.

(2) Evaluation of enzyme combination in saccharifying enzyme-secreting yeast culture supernatant solution (Part 1)
As the ionic liquid treatment, 1-Butyl-3-methylimidazolium acetate ([Bmim] [OAc]) was used. That is, 1.0 g of ionic liquid was collected in a vial, and 50 mg of a biomass sample was added thereto. As the biomass, bagasse powder adjusted to a particle size of about 250 μm that was crushed by a cutter mill was used. This was treated at 120 ° C. for 30 minutes, and washed with 9 ml of sterilized water using a filter membrane. After adding 1 ml of 100 mM citrate buffer (pH 5.0) to the treated sample, add 1 ml each of the saccharifying enzyme-secreting yeast culture supernatant solution constructed in Example 1 above, and use 10 ml of sterilized water for a total volume of 10 ml. It was.

  For preparation of the culture supernatant solution, each genetically modified yeast constructed in Example 1 above was cultured with shaking in 5 ml of YPD culture at 30 ° C. for 63 hours, and the resulting cells were collected. Thus, a culture supernatant solution was obtained. This solution was subjected to a commonly reported cellulose degradability test, and 1 ml of a saccharifying enzyme-secreting yeast culture supernatant solution in which cellulolytic activity was confirmed was used.

  When adding yeast supernatant solution to ionic liquid-treated bagasse, the seven cellulase-secreting strains constructed this time (two types of cellobiohydrase type I saccharifying enzymes, hereinafter referred to as CBHI, two types of cellobiohydrase type II) Eight possible combinations of saccharifying enzymes, hereinafter referred to as CBHII, two types of endoglucanase type I saccharifying enzymes, hereinafter referred to as EGI, one type of betaglucosidase saccharifying enzyme, hereinafter referred to as BGL, were carried out (see Table 2). ).

  As a control for this test, a commercially available cellulolytic enzyme was used. This cellulase mixed solution was prepared by mixing Novozyme-Celluclast (Sigma-Aldrich) consisting of Trichoderma reesei ATCC 26921 and Novozyme 188 (Sigma-Aldrich) consisting of Aspergillus niger at a ratio of 5: 1 to 6 FPU / g biomass. It added so that it might become.

Sampling was performed after 0, 3, 8, 24, 48, 72 hours from the sample of the saccharification reaction, and the glucose concentration in the solution was measured. Biosensor BF-5 (Oji Scientific Instruments) was used for measuring the glucose concentration, and the details of the operation were in accordance with the attached protocol. Based on the obtained glucose concentration, the conversion efficiency to sugar when the cellulose contained in each biomass was 100 was calculated according to the following formula.
Glucose conversion efficiency (%) = [produced glucose] / [glucose units in cellulose] ´ 100

  FIG. 3 shows the change over time in the saccharification rate and the saccharification effect for each combination. As shown in FIG. 3, it was confirmed that the combination of Clostridium thermocellum-derived CBHA, Phanerochaete chrysosporium-derived CBH II, Trichoderma reesei-derived EGII, and Aspergillus aculeatus-derived BGL showed high saccharification efficiency. This combination was named Best Mix Ver.1. The cellulose saccharification efficiency with the addition of a commercially available enzyme (6 FPU / g biomass) in this test was 79.6%.

(3) Evaluation of effect of preferred combination (Best mix ver.1) in the difference in biomass pretreatment (Best mix ver.1) CBHA derived from Clostridium thermocellum, CBH II derived from Phanerochaete chrysosporium, Trichoderma reesei-derived EG II and Aspergillus aculeatus-derived BGL) were compared with other biomass pretreatment effects. In addition, the dilute sulfuric acid process known as a general pretreatment method was implemented.

  Moreover, from the result of Example 2, as a negative combination (Negative Mix) that does not show high saccharification efficiency, a mixed solution of Cel9K derived from Clostridium thermocellum, CBH II derived from Trichoderma reesei, Cel8C derived from Clostridium cellloyticum, and BGL derived from Aspergillus aculeatus is similarly applied. The test was conducted.

  As the ionic liquid treatment, [Bmim] [OAc] was used and the same treatment as in Example 2 was performed.

For dilute sulfuric acid treatment, 1 ml of H ₂ SO ₄ 2% (wt./vol.) Was collected in a vial according to a general method, and 50 mg of a biomass sample was added thereto. As the biomass, bagasse powder adjusted to a particle size of about 250 μm that was crushed by a cutter mill was used. This was autoclaved at 120 ° C. for 90 minutes, and washed with 9 ml of sterilized water using a filter membrane. After adding 1 ml of 100 mM citrate buffer (pH 5.0) to the treated sample, add 1 ml each of the saccharifying enzyme-secreting yeast culture supernatant solution constructed in Example 1 above, and use 10 ml of sterilized water for a total volume of 10 ml. It was.

  For the preparation of the culture supernatant solution, each genetically modified yeast identified in Example 2 above was shaken and cultured in 5 ml of YPD culture solution at 30 ° C. for 17 hours, and the resulting cells were collected. Thus, a culture supernatant solution was obtained. This solution was subjected to a commonly reported cellulose degradability test, and 1 ml of a saccharifying enzyme-secreting yeast culture supernatant solution in which cellulolytic activity was confirmed was used.

  The control for this test was prepared in the same manner as in Example 2.

  Sampling was performed after 24 and 48 hours from the sample of the saccharification reaction, and the glucose concentration in the solution was measured in the same manner as in Example 2 to calculate the conversion efficiency to sugar. The cellulose saccharification efficiency of bagasse due to the difference in pretreatment is shown in FIG.

  As shown in FIG. 4, the preferred saccharifying enzyme combination (Best mix ver. 1; CBHA derived from Clostridium thermocellum, CBH II derived from Phanerochaete chrysosporium, EG II derived from Trichoderma reesei, BGL derived from Aspergillus aculeatus) The improvement effect was 5 times higher than this, and this value was much higher than the double effect, which is the difference between the two in dilute sulfuric acid treatment. In addition, saccharification efficiency per se was confirmed to be more than double that of [Bmim] [OAc] treatment compared to dilute sulfuric acid treatment. From this, it was confirmed that this preferred saccharifying enzyme combination exhibits a high saccharifying effect particularly in ionic liquid treatment.

(4) Construction of saccharogenic enzyme-producing yeast (part 2)
In order to further enhance the cellulose saccharification efficiency, the preferred combination obtained in Example 2 (Best mix ver. 1; CBHA derived from Clostridium thermocellum, CBH II derived from Phanerochaete chrysosporium, EG II derived from Trichoderma reesei, BGL derived from Aspergillus aculeatus) New saccharifying enzymes were selected. In implementation, five new saccharogenic enzyme-producing yeasts were constructed.

The newly added saccharifying enzymes were five saccharifying enzymes shown in the following table. Among them, Trichoderma reesei-derived EG II and Trichoderma reesei-derived Xylanase II (Xyn II) were designed to fit yeast codon usage and newly synthesized genes were used. The nucleotide sequences of both newly synthesized genes (Trichoderma reesei-derived EG II, Trichoderma reesei-derived Xylanase II) are represented by SEQ ID NO: 23 and SEQ ID NO: 24. For the remaining 3 types of Endoglucanase III (EG III), genes were obtained by PCR. The obtained gene fragment was introduced into the pAUR112-GAPSSRG vector (FIG. 2) by In Fusion PCR Cloning (Clontech). The ligation solution was transformed into E. coli (ECOS ^™ Competent E. coli DH5α; Nippon Gene) and each clone grown on an LB plate supplemented with ampicillin to a final concentration of 50 μg / ml was subjected to colony PCR. A clone was selected. The target clone was cultured in an LB culture solution containing ampicillin, plasmid DNA was extracted from the main culture solution, and this mixed DNA solution was used in the subsequent experiments. Moreover, it was confirmed by the gene sequence analyzer ABI PRISM 310 Genetic Analyzer (Applied Biosystems) whether it was the target vector.

  Transformation into host yeast and selection were carried out in the same manner as in Example 1. The selected genetically modified yeast was confirmed for the presence of the target fragment in the same manner as in Example 1.

(5) Evaluation of enzyme combination in saccharifying enzyme-secreting yeast culture supernatant solution (Part 2)
In order to further enhance the cellulose saccharification efficiency, the preferred combination obtained in Example 2 (Best mix ver. 1; CBHA derived from Clostridium thermocellum, CBH II derived from Phanerochaete chrysosporium, EG II derived from Trichoderma reesei, BGL derived from Aspergillus aculeatus) Using the saccharogenic enzyme-secreting yeast supernatant solution constructed in Example 4, a new saccharifying enzyme suitable combination was examined for ionic liquid-treated biomass.

  As the ionic liquid treatment, [Bmim] [OAc] was used and the same treatment as in Example 2 was performed. For the preparation of the culture supernatant solution, each genetically modified yeast constructed in Example 4 was treated in the same manner as in Example 2, and 1 ml of cellulase-secreting yeast culture supernatant solution in which saccharifying enzyme-degrading activity was confirmed. It was used. The control for this test was prepared in the same manner as in Example 2. Sampling was performed after 0, 24, 48, 72 hours from the saccharification reaction sample, and the glucose concentration was measured in the same manner as in Example 2 to calculate the conversion efficiency to sugar. FIG. 5 shows changes with time in the saccharification rate for each combination of enzymes.

  As shown in FIG. 5, in the combination of Trichoderma reesei Xylanase (codon changed to yeast), Trichoderma reesei EG II (codon changed to yeast), Clostridium thermocellum CBHA, Phanerochaete chrysosporium CBH II, Aspergillus aculeatus BGL It was confirmed that the saccharification efficiency was close to that of cellulose saccharification with the addition of a commercially available enzyme (6 FPU / g biomass). This preferred saccharifying enzyme combination was named Best Mix Ver.2. On the other hand, in the test system to which three kinds of endoglucanases (EG III) were added, no significant saccharification improvement effect was observed.

(6) Simultaneous saccharification and fermentation evaluation using a suitable saccharifying enzyme combination Subsequently, a saccharification and fermentation test using the above-described suitable saccharifying enzyme combination was attempted.
[Bmim] [OAc] was used as the ionic liquid. Ionic liquid 2.0 g was collected in a vial and 100 mg biomass sample was added to it. As the biomass, bagasse powder having a particle size of 250 μm, which was crushed by a cutter mill, was used. This was treated at 120 ° C. for 30 minutes, and washed with 18 ml of sterilized water using a filter membrane. The treated sample was 2 ml of 5 × YP medium, 1 ml of ampicillin (final concentration 50 μg / ml), and a suitable saccharifying enzyme combination identified in Example 5 (Trichoderma reesei-derived Xylanase, Trichoderma reesei-derived EG II, Clostridium Thermocellum-derived CBHA, Phanerochaete chrysosporium-derived CBH II, and Aspergillus aculeatus-derived BGL) were added to each of the mixed solutions of the yeast and the culture supernatant to prepare a total volume of 10 ml with sterile water.

  In this test, a sample prepared by mixing non-recombinant yeast with a commercially available cellulolytic enzyme was used as a positive control. This cellulase mixed solution was prepared by mixing Novozyme-Celluclast (Sigma-Aldrich) composed of Treichoderma reesei ATCC 26921 and Novozyme 188 (Sigma-Aldrich) composed of Aspergillus niger in a ratio of 5: 1 to 6 FPU / g biomass. It added so that it might become. As a negative control, samples without yeast strain were also prepared.

  The simultaneous saccharification and fermentation conditions were carried out at 35 ° C. and 300 rpm. Sampling was performed at regular intervals, and the ethanol concentration in the solution was measured. Biosensor BF-5 (Oji Scientific Instruments) was used for measurement, and the details of the operation followed the attached protocol. Based on the obtained ethanol concentration, the ethanol conversion efficiency when the cellulose contained in each biomass was taken as 100 was calculated. The result is shown in FIG. As shown in FIG. 6, in the test section where a culture medium of a suitable combination of five types of saccharifying enzymes identified by the current selection and yeast were added, a maximum of 50%, although no commercially available enzyme was added. A degree of ethanol production yield was confirmed.

Claims (15)

A method for producing useful substances,
Contacting the cellulose-containing material with the ionic liquid to infiltrate the cellulose-containing material with the ionic liquid;
Contacting the solid phase containing the cellulose-containing material with an enzyme group containing cellulase and xylanase, and decomposing cellulose in the solid phase;
Producing a useful substance by fermentation of microorganisms using a carbon source containing a cellulose degradation product obtained in the cellulose degradation step;
A production method comprising:   The enzyme group includes one or both of Clostridium thermocellum cellobiohydrolase I (CtCel9A) and Clostridium thermocellum cellobiohydrolase I (CtCbhA), Phanerochaete chrysosporium cellobiohydrolase II (PcCBH II), Trichoderma reesei The production method of Claim 1 containing the endoglucanase II (TrEG II) derived from xylanase II (TrXyn II) derived from Trichoderma reesei.   The production method according to claim 1 or 2, wherein the enzyme group includes cellobiohydrolase I (CtCbhA) derived from Clostridium thermocellum.   The production method according to any one of claims 1 to 3, wherein the enzyme group further contains β-glucosidase (AaBGA) derived from Aspergillus aculeatus.   The production method according to claim 1, wherein the cellulose-containing material contains crystalline cellulose.   The production method according to claim 1, further comprising a step of solid-liquid separation of the solid phase and the ionic liquid prior to the decomposition step.   The production method according to any one of claims 1 to 6, wherein at least a part of the enzyme of the enzyme group is supplied by the microorganism being presented on a cell surface layer or secreted outside the cell.   The production method according to claim 7, wherein all the enzymes in the enzyme group are supplied by the microorganisms presenting on the cell surface layer.   The production method according to claim 1, wherein the ionic liquid contains 1-butyl-3-methylimidazolium acetate.   Furthermore, the production method in any one of Claims 1-9 provided with the process of supplying the said ionic liquid isolate | separated at the said solid-liquid separation process to the said osmosis | permeation process. An enzyme preparation for degrading cellulose-containing material,
One or both of cellobiohydrolase I (CtCel9A) from Clostridium thermocellum and cellobiohydrolase I (CtCbhA) from Clostridium thermocellum, cellobiohydrolase II (PcCBH II) from Phanerochaete chrysosporium, endoglucanase from Trichoderma reesei (TrEG II) and an enzyme preparation containing Trichoderma reesei-derived xylanase II (TrXyn II).   The enzyme preparation according to claim 11, comprising cellobiohydrolase I (CtCbhA) derived from Clostridium thermocellum.   The enzyme preparation according to claim 11 or 12, comprising β-glucosidase (AaBGA) derived from Aspergillus aculeatus. A microbial preparation for degrading a cellulose-containing material,
One or both of cellobiohydrolase I (CtCel9A) from Clostridium thermocellum and cellobiohydrolase I (CtCbhA) from Clostridium thermocellum, cellobiohydrolase II (PcCBH II) from Phanerochaete chrysosporium, endoglucanase from Trichoderma reesei (TrEG II), and Trichoderma reesei-derived xylanase II (TrXyn II), including one or more microorganisms that present or secrete one or more of these enzymes to the cell surface, Microbial preparation. A method for producing a cellulose degradation product,
Contacting the cellulose-containing material with the ionic liquid to infiltrate the cellulose-containing material with the ionic liquid;
Contacting the solid phase containing the cellulose-containing material with an enzyme group containing cellulase and xylanase, and decomposing cellulose in the solid phase;
A method comprising: