JP4431106B2 - Method for producing cellooligosaccharide - Google Patents

Description

  The present invention relates to a method for producing cellooligosaccharide by allowing cellulase to act on cellulose or a cellulose-containing substance.

  In recent years, due to an increase in health consciousness, it is desired to provide oligosaccharides having functionality such as bifido activity. Cellobiose, which is the main component of the cellooligosaccharide produced by the present invention, is a disaccharide in which two glucoses are linked by β-1,4, and is known as a constituent oligosaccharide of cellulose. Cellobiose is an enzyme degradation product of cellulose that has been used for a long time as a food, and is a natural product that is also present in corn stalks, so there is no problem in safety. It is also known that it does not decompose at all in the human stomach and small intestine, reaches the large intestine, and is selectively assimilated by short-chain fatty acid-producing bacteria such as butyric acid bacteria in the large intestine to produce short-chain fatty acids. ing. In recent years, various physiological functions of short chain fatty acids have attracted attention.

Cellooligosaccharides such as cellobiose are components of cellulose, which is biomass produced in large quantities in nature, but have not been industrially produced so far. One reason is that cellulase is generally a complex enzyme system, which acts on cellulose to produce cellooligosaccharide (mainly endoglucanase, cellobiohydrase), and enzyme that breaks down the produced cellobiose into glucose (β- The presence of this β-glucosidase significantly reduces the yield of cellooligosaccharide. The second cause is that the substrate cellulose is insoluble and there is a region that is highly resistant to the enzyme called the crystalline region, making it difficult for the enzyme reaction to proceed efficiently, and the generated cellobiose is due to feedback inhibition. This is because there are various difficult problems such as inhibiting the cellulose decomposition reaction by cellulase.
Under such circumstances, many attempts have been made to overcome the above problems. First, an attempt to eliminate the influence of β-glucosidase contained in cellulase is shown below.

  Method for selectively producing cellobiose from cellulose using cellulase from which β-glucosidase present in cellulase is removed by adsorbing cellulase to a weakly acidic cation exchange resin equilibrated to pH 3.5 to pH 5.0 (Patent Document 1), a method of producing cellobiose by coexisting δ-gluconolactone or gluconic acid, which is an inhibitor of β-glucosidase, in the decomposition of cellulose by cellulase (Patent Document 2), decomposition of cellulose by cellulase In this process, glucose oxidase is allowed to coexist to produce δ-gluconolactone from the produced glucose, and as a result, β-glucosidase activity is suppressed (Patent Document 3), and cellulase is added to insoluble cellulose for 30-60. After incubating in an aqueous medium at ℃ for 1-4 hours, decompose A method of separating the solid content in the water-soluble medium before completion of the saccharification reaction, adding a water-soluble solution to the solid content without adding a new substrate, and heating for a certain period of time to collect the cellobiose produced in the aqueous solution ( Patent document 4) etc. are mentioned.

  However, these methods require cost and labor to remove β-glucosidase, so it is not practical, and the method of coexisting with δ-gluconolactone is complicated to separate from the product cellobiose, Since δ-gluconolactone is expensive, practical application has been difficult. Moreover, in the method of adsorbing an enzyme to cellulose, cellobiose is generated only from the cellulose added first, there is a problem that the yield is poor and continuous industrial production is not possible.

On the other hand, in order to eliminate the influence of β-glucosidase, in addition to inhibiting the enzyme, a method of extracting the produced cellobiose out of the reaction system has been studied.
As an example, a method in which a reaction vessel in which cellulose and cellulase coexist and an ultrafiltration membrane are combined, and only the produced cellobiose is selectively extracted (Non-Patent Document 1) and the like can be mentioned.
However, in the method of collecting cellobiose using an ultrafiltration membrane, there are problems such that the apparatus becomes complicated and the ultrafiltration membrane is clogged by a cellulose degradation product.

  In addition, regarding the improvement of the enzyme reactivity with respect to cellulose as a substrate, a method using slush pulp not subjected to a drying step as a raw material (Patent Document 5), pretreatment of cellulose, crystallinity and water retention are specified. Examples include a method using a raw material adjusted within the value (Patent Document 6).

JP-A-5-115293 JP-A-4-75594 JP-A-8-33496 Japanese Patent Publication No. 6-83684 Japanese Patent Publication No. 8-2312 JP 2005-68140 A Nippon Nogeikagaku Kuishi (Vol. 63, No. 6, 1989 p1133-1135)

  The object of the present invention is to reduce the amount of glucose as a by-product when producing cellooligosaccharides that are expected as functional oligosaccharides from cellulose or cellulose-containing substances, and at low cost, easily and efficiently It is in providing the manufacturing method of.

  As a result of intensive studies to solve these problems, the present inventors have continuously added the substrate cellulose or the cellulose-containing substance and the aqueous medium to the reaction vessel in which the cellulase and the substrate cellulose or the cellulose-containing substance coexist. Alternatively, it was found that cellooligosaccharides can be produced efficiently by continuously or sequentially withdrawing the reaction saccharified solution while being added sequentially, and the present invention has been completed. In addition, it has been found that cellooligosaccharides can be efficiently produced by using cellulose or cellulose-containing substances in which only the enzyme components necessary for cellooligosaccharide production are adsorbed in advance in the enzyme components contained in cellulase as the reaction substrate. The present invention has been completed.

That is, the present invention provides the following [1] to [6].
[1] In a reaction system in which cellulase is reacted with cellulose or a cellulose-containing substance to produce cellooligosaccharide, cellulose or cellulose-containing substance and an aqueous medium are continuously added or sequentially added to the reaction system, while continuously or sequentially reacting. A method for producing a cellooligosaccharide, which comprises extracting a reaction solution out of the system.
[2] The reaction solution extracted from the reaction system is separated into a reaction saccharification solution and cellulose or cellulose-containing substance, and then the separated cellulose or cellulose-containing substance is returned to the reaction system. [1] A process for producing a cellooligosaccharide according to [1].
[3] It is characterized in that an enzyme component other than β-glucosidase among the enzyme components contained in cellulase is adsorbed in advance to the cellulose or cellulose-containing substance added in the initial stage and / or during the reaction in the reaction system. The method for producing a cellooligosaccharide according to [1] or [2].
[4] When adsorbing enzyme components other than β-glucosidase among cellulose components or cellulose-containing substances in advance, the pH is adjusted to 5.0 to 10.0 and the slurry concentration is adjusted to 1 to 10% by weight. The method for producing a cellooligosaccharide according to the above [3], wherein the cellulose slurry is treated by adding 5 to 300% by weight of cellulase to the cellulose.
[5] The method for producing a cellooligosaccharide according to any one of [1] to [4], wherein the cellulose concentration in the reaction system is maintained at 1% by weight or more.
[6] The above-mentioned [1] to [5], wherein as the cellulose or cellulose-containing substance, pulp subjected to at least one of fibrillation treatment, mechanochemical treatment, and chemical treatment is used. The manufacturing method of the cellooligosaccharide as described in any one.

  According to the present invention, when producing cellooligosaccharides from cellulose or a cellulose-containing substance, the production of cellooligosaccharides can be efficiently suppressed by suppressing the amount of glucose produced without inhibiting the β-glucosidase activity contained in cellulase. I can do it. In addition, the reaction solution extracted from the reaction system contains almost no enzyme components contained in cellulase other than β-glucosidase, so it is not necessary to separate and recover only the enzyme with an ultrafiltration membrane, and it is inexpensive and easy. Cellooligosaccharides can be produced. In addition, since it is not necessary to use an ultrafiltration membrane that tends to cause clogging such as clogging in the reaction system, cellooligosaccharides can be produced continuously and efficiently industrially.

  The cellulase used in the present invention is not limited as long as it decomposes cellulose into cellooligosaccharide (polysaccharide in which 2 to 10 or more D-glucopyranoses are linked by β-1,4 bonds), and its origin is limited. However, cellulases such as filamentous fungi, basidiomycetes, and bacteria can be used. Illustratively, Trichoderma genus, Aspergillus genus, Irpex genus, Aeromonas genus, Clostridium genus, Bacillus genus, Pseudomonas genus, Penicillium genus, Humicola genus and / or produced by genetic recombination alone Alternatively, two or more kinds can be mixed and used. In addition, as the enzyme source, commercially available cellulase preparations, cultures of the above-mentioned bacteria, and filtrates thereof can also be used directly. Among them, a cellulase having a strong cellulose decomposing power such as a cellulase derived from the genus Trichoderma or Aspergillus is preferable.

  Examples of commercially available enzymes include Mecellase (manufactured by Meiji Seika Co., Ltd.), cell crust (manufactured by Novozymes), cellulase Amano A (manufactured by Amano Enzyme), cellulase Amano T (manufactured by Amano Enzyme), Cellulase Daiwa (manufactured by Daiwa Kasei Co., Ltd.), cell riser (manufactured by Nagase Seikagaku Co., Ltd.), doriserase (manufactured by Kyowa Hakko Co., Ltd.), cellulosin (manufactured by Hankyu Bio Industry Co., Ltd.), etc. can be used. .

  Cellulose or a cellulose-containing substance (hereinafter sometimes simply referred to as a substrate) used as a substrate means cellulose itself or a substance containing cellulose as a component. Cellulose may be water-insoluble or water-soluble, but a raw material close to natural cellulose, i.e., unmodified and water-insoluble cellulose is preferable, and in the case of a cellulose-containing substance, it contains a large amount of unmodified and water-insoluble cellulose. Is preferable because the production efficiency of cellooligosaccharide is high.

  As the water-insoluble cellulose, linter pulp, kraft pulp, sulfite pulp, dissolved pulp and the like are preferable in terms of price and purity, and these may be dried or not dried. Dissolving pulp means pulp specially refined chemically and used mainly by dissolving in chemicals. Dissolving pulp is produced by taking out highly pure cellulose from wood. That is, wood contains three major components of cellulose, lignin, and hemicellulose, and a small amount of resin and ash. First, most of the lignin and hemicellulose are removed by cooking, and then remain in the subsequent purification and bleaching stages. Remove lignin, hemicellulose, resin and ash. Dissolving pulp is used as a raw material for chemical fibers, cellophane, plastics, synthetic pastes, and various other cellulosic derivatives.

  Undried pulp (slush pulp) includes bleached or unbleached kraft slush wood pulp, bleached or unbleached sulfite slush wood pulp, bleached or unbleached dissolved slush wood pulp, bleached or unbleached kraft slash non-wood pulp, bleached Alternatively, unbleached sulfite slush non-wood pulp, bleached or unbleached dissolved slush non-wood pulp, crude or refined slush linter pulp can be used. It should be noted that the removal of lignin in the pulp increases the pore volume, and the removal of hemicellulose and the like increases the pure cellulose content. Wood pulp, bleach-dissolved slush non-wood pulp, and refined slush linter pulp are preferred.

  Moreover, although the water | moisture content contained in a slush pulp is influenced by pulp manufacturing conditions, ie, the conditions of a dehydration process, it is desirable that it is 50 to 99 weight%, More preferably, it is 60 to 98 weight%. When the water content is less than 50% by weight, the pore volume in the pulp may be crushed and keratinized due to the share generated by the dehydration process, which may cause a decrease in accessibility of cellulose. If it exceeds wt%, when it is added to the reaction system as a substrate, the pulp concentration in the reaction system becomes too low, and it is necessary to remove moisture such as dehydration before starting the reaction. It becomes complicated and is not preferable.

  Dry pulp (dry pulp) includes bleached or unbleached kraft wood pulp, bleached or unbleached sulfite wood pulp, bleached or unbleached dissolved wood pulp, bleached or unbleached kraft non-wood pulp, bleached or unbleached sulfite Wood pulp, bleached or unbleached dissolved non-wood pulp, crude or refined linter pulp, waste paper pulp, deinked pulp, cellulose produced by microorganisms such as squirt cellulose and acetic acid bacteria, or the above-mentioned cellulosic materials with caustic soda solution, copper It is possible to use regenerated cellulose which has been dissolved in some solvent such as ammonia solution or morpholine derivative and then spun again, mercerized pulp treated with caustic soda solution, or the like. In addition, since the cellulose pure content becomes high by removing the lignin content in the pulp and removing hemicellulose and the like, among the illustrated cellulose materials, bleach-dissolved wood pulp, bleach-dissolved non-wood pulp Refined linter pulp, regenerated cellulose, and mercerized pulp are preferred.

  The moisture contained in the dry pulp depends on the pulp production conditions, that is, the drying process conditions, but is preferably 5 to 30% by weight. When the water content is less than 5% by weight, the pulp becomes keratinized, that is, hydrogen bonds between molecules and molecules may progress, and the accessibility of cellulose may decrease. On the other hand, when the water content exceeds 30% by weight Is not preferred because of high transportation costs and rot. In addition, cellulose-containing substances including cellulose such as wood flour, building waste, and agricultural waste can be used as a substrate.

  The above-mentioned cellulose or cellulose-containing substance generally has a crystalline portion that is resistant to degradation by enzymes. Therefore, this crystal structure is broken or changed, the surface area of cellulose is increased, and cellulase is easily adsorbed on the cellulose surface. By using the product that has been subjected to the treatment, the reactivity to cellulase is increased, and the production rate of cellooligosaccharide can be increased. Examples of such treatment include chemical treatment such as acid, alkali, and ozone treatment, mechanical treatment such as explosion treatment, fibrillation treatment, and pulverization treatment, radiation treatment such as electron beam and γ-ray, and mechanochemical treatment. It is done.

  Treatments that break or change the crystal structure include mainly mechanochemical treatments and / or chemical treatments. Treatments that increase the surface area of cellulose and facilitate the adsorption of cellulase to the cellulose surface are mainly fibrillated. There is processing. The treatment can be appropriately selected from these treatments, and the reactivity of cellulose can be improved more efficiently by combining a plurality of treatments than one treatment.

The change in cellulose crystal structure of various pulps can be measured by an index called cellulose I-type crystallinity, and the specific surface area of cellulose can be measured by an index called water retention. The lower the cellulose I-type crystallinity, Higher is better. If the water retention is low, the swelling of the pulp and the fluffing of the fiber surface are reduced, and the surface area of the cellulose is reduced, which leads to a decrease in the accessibility of the cellulose.
In this specification, the water retention is J. It is defined by TAPPI NO 26-78, and indicates the percentage of water held by pulp relative to absolutely dry pulp after centrifuging (3000 G) a certain amount of pulp suspension. The higher the water retention, the lower the interfiber binding force and the more water contained in the fiber, that is, the cellulose surface area tends to increase.

  If the cellulose has a high cellulose I-type crystallinity, the structural change of the cellulose in the pulp before and after the pretreatment is small, which is not preferable because accessibility is not improved. In the present specification, cellulose type I means a crystalline form of natural cellulose. The crystallinity means the ratio of the amount of crystal region of cellulose to the total amount, and can be measured by X-ray diffraction method or the like. The degree of crystallinity is also related to the physical and chemical properties of cellulose, and the larger the value, the higher the crystallinity of cellulose and the less non-crystalline parts, so the hardness, density, etc. increase, but elongation, flexibility, Accessibility and chemical reactivity to water and solvents are reduced.

  The fibrillation treatment uses a machine that grinds and shears pulp for the purpose of increasing the surface area of cellulose and facilitating the adsorption of cellulase to the cellulose surface. (1) Fine structure of cellulose fibers (2) make the surface of the cellulose fiber fluffy, or branch the fiber to increase the fiber surface area (external fibrillation), (3) fine fiber And consequently increase the fiber surface area.

For the fibrillation treatment, a known model may be used as long as it can be fibrillated by grinding and shearing cellulose fibers under wet conditions. For example, beaters (Niagara Beater Kumagaya Rikyu Kogyo), PFI mills (PFI Mill Kumagaya Rikyu Kogyo), refiners (Disk Refiner Tozai Seiki Co.), and high-pressure homogenizers (Niro)・ Soabi), ultra-high pressure homogenizer (Shinmaru Enterprises), Nanomizer (Special Machinery), and stone mill (supermass colloider, Serendipeater are both made by Masuyuki Sangyo).
In the present invention, the yield of cellooligosaccharide can be increased by using as a substrate a cellulose or a cellulose-containing substance that has been subjected to a combination of alkali treatment and fibrillation treatment.

Hereinafter, the fibrillation treatment when various pulps are used as the substrate of the present invention will be described in detail.
The pulp concentration during the fibrillation treatment is generally 1.0 to 20% by weight, and more preferably 2.0 to 15% by weight. When the pulp concentration is less than 1.0% by weight, not only the treatment amount is remarkably increased, but also water acts as a plasticizer and the degree of fibrillation treatment is reduced, and the pulp concentration exceeds 20% by weight. In such a case, the pulp concentration is too high, the fluidity is lowered, and it becomes difficult to perform the fibrillation treatment. The fibrillation time is preferably 3 minutes to 5 hours, more preferably 5 minutes to 3 hours, although the optimum value varies depending on the pulp concentration and the model used. When the fibrillation treatment time is less than 3 minutes, there is little change in the shape of the pulp, and there is little increase in the water retention, and when the treatment time exceeds 5 hours, the cellulase reaction efficiency and the pulp treatment cost are reduced. In comparison, the effect is remarkably reduced, which is not preferable. In addition, in the model which performs a continuous process like a stone mill type grinder, it can process repeatedly or repeatedly in multiple times so that it may become said process time.

The mechanochemical treatment of the present invention is performed for the purpose of lowering the crystallinity of the cellulose fiber, and by applying strong energy to the cellulose fiber, the crystal structure of the cellulose itself is changed, that is, the crystallinity is lowered. It is to let you.
As long as the mechanochemical treatment is a treatment capable of reducing the crystallinity of cellulose, a known method may be used, but a treatment using a medium mill is preferable. As the media mill, a known model may be used. If the model is purposely exemplified, a vibration ball mill (manufactured by Chuo Kako), an S / G mill (manufactured by Igarashi Machine), an apex mill (manufactured by Kotobuki Giken Kogyo), a super apex. Examples include mills (manufactured by Kotobuki Giken Kogyo), aquamizers (manufactured by Hosokawa Micron), and ultra visco mills (manufactured by IMEX).

The mechanochemical treatment when various pulps are used as the substrate of the present invention will be described in detail below.
The mechanochemical treatment is performed in a state where 100 parts of pulp is dispersed in 0 to 10,000 parts of water, more preferably in 0 to 5000 parts of water. When the amount of water exceeds 10,000 parts, not only the treatment amount is remarkably increased, but also water acts as a plasticizer and the mechanochemical treatment is lowered, which is not preferable.

  As the medium of the medium mill used for the mechanochemical treatment, commercially available alumina, ceramic, titanium, zirconia, glass, etc. having a diameter of 1 mm to 50 mm can be used, and the filling rate is 50% by volume to 95%. Volume% is preferred. When the filling rate is less than 50% by volume, the contact frequency with respect to cellulose is decreased, and mechanochemical reaction is less likely to occur, and when the filling rate exceeds 95% by volume, the movement of the medium is hindered. Absent.

  The treatment time is preferably 5 minutes to 5 hours, more preferably 10 minutes to 3 hours. In the present invention, the time for mechanochemical treatment is important. When the treatment time is less than 5 minutes, almost no change is observed in the crystal structure of cellulose, and when it exceeds 5 hours, the cellulose I-type crystallinity decreases and the crystal structure becomes amorphous, but the cellulose fiber Is agglomerated and hardened, which is not preferable because the water retention is significantly reduced. In general, there is a correlation between the water retention and the surface area of cellulose, and when the water retention decreases, the surface area decreases. Therefore, even if the crystallinity decreases, the reactivity of cellulase decreases.

In the present invention, the chemical treatment is aimed at lowering the crystallinity of cellulose, and alkali treatment represented by mercerization treatment is preferred.
The alkali treatment when various pulps are used as the substrate of the present invention will be described in detail below.

  As the alkali treatment, it is preferable to adjust the pulp concentration to 1.0 to 20% by weight and adjust the caustic soda concentration to 3 to 30% by weight, and to treat for 5 minutes to 5 hours. More preferably, the pulp concentration is adjusted to 2.0 to 15% by weight and the caustic soda concentration is adjusted to 5 to 20% by weight, and the treatment is performed for 10 minutes to 3 hours.

  When the pulp concentration is less than 1.0% by weight, not only the processing amount is remarkably increased, but also the caustic soda amount is increased in order to maintain the caustic soda concentration, that is, the chemical cost is greatly increased. If it exceeds 20% by weight, the pulp concentration is too high, the fluidity is lowered, and the alkalinization treatment proceeds non-uniformly, which is not preferable.

  When the caustic soda concentration is less than 3% by weight, no change in the crystal structure of the cellulose is observed, and when the caustic soda concentration exceeds 30% by weight, the cellulose I-type crystals have already completely disappeared and become amorphous. An excessively high concentration treatment is not preferable because it leads to a significant increase in chemical cost.

  When the alkali treatment time is shorter than 5 minutes, the progress of the mercerization reaction is small, and the degree of decrease in the cellulose I-type crystallinity is small. Further, even if the alkali treatment time exceeds 5 hours, the further crystal structure of cellulose Since the change of is not seen, it is not preferable.

  Examples of cellulose soluble in water include, but are not limited to, cellulose derivatives (modified cellulose) such as sodium carboxymethylcellulose, methylcellulose, and hydroxyethylcellulose. In addition, when water-soluble cellulose is used as a raw material, it is poured into a water-containing solvent such as methanol, ethanol, propanol, isopropanol, acetone or the like so that only the cellulose is precipitated, and the insoluble cellulose is recovered. The produced cellooligosaccharide can be separated.

  A feature of the present invention is that in a reaction system in which cellulose or a cellulose-containing substance and cellulase are reacted in an aqueous medium, cellulose or the cellulose-containing substance and the aqueous medium are added continuously or sequentially, while cellooligosaccharide as a reaction product is added. The reaction is performed by continuously or sequentially extracting a certain amount of the reaction solution. This focuses on the property that enzyme components other than β-glucosidase in cellulase are adsorbed to cellulose, aiming to leave enzyme components other than β-glucosidase in the reaction system and to discharge only β-glucosidase out of the system. It is a thing. In general, a cellulase mainly contains three kinds of enzyme components such as endoglucanase, cellobiohydrase, and β-glucosidase. Of these, endoglucanase and cellobiohydrase are involved in the production of cellooligosaccharide, and β-glucosidase is an enzyme component that decomposes the produced cellooligosaccharide into glucose. Therefore, in order to produce cellooligosaccharides, it is better that less β-glucosidase is present in the reaction system.

  In this specification, the reaction liquid means a mixture of cellulose or cellulose-containing substance, aqueous medium and reaction product present in the reaction system, and the reaction saccharified liquid is obtained by removing cellulose or cellulose-containing substance from the reaction liquid. Means things.

The outline of the reaction system of the main aspect of the present invention is shown in FIGS.
In the present invention, at the initial stage of the reaction, the produced cellooligosaccharide is decomposed by β-glucosidase contained in cellulase, so the production rate of cellooligosaccharide (cellobiose production / glucose production) is lowered. However, since free β-glucosidase that is not adsorbed to cellulose in the reaction solution is removed from the system by extracting a certain amount of the reaction solution, the amount of β-glucosidase remaining in the reaction system decreases with time. Thus, the conversion of cellooligosaccharide to glucose is suppressed, and as a result, the production rate of cellooligosaccharide increases.

  In the present invention, the aqueous medium used in the reaction system is preferably a buffer solution such as an acetate buffer, ion-exchanged water or the like, but tap water or the like can also be used sufficiently.

  The cellulose concentration in the reaction system needs to be maintained at about 1 to 10% by weight, preferably about 1 to 7% by weight while the reaction is continued. When the cellulose concentration is less than 1% by weight, the amount of cellooligosaccharides is reduced because the amount of substrate is too small, and the work required for post-treatment such as concentration increases, and when the cellulose concentration exceeds 10% by weight, the reaction This is not preferable because the viscosity in the system becomes too high, workability deteriorates, and the enzyme reaction rate also decreases. When a cellulose-containing substance is used as a substrate, the cellulose concentration in the reaction system is adjusted to fall within the above range according to the content of cellulose contained therein. Moreover, when adding cellulose in the middle of the reaction, the amount of cellulose remaining in the reaction system is measured and adjusted so that the cellulose concentration in the system falls within the above range. The addition amount is preferably about 0.3 to 2.0 times that of the saccharified substrate. Similarly, when returning the cellulose contained in the reaction liquid extracted from the reaction system to the system again (in the case of FIGS. 1 and 2, to the reaction tank), the cellulose concentration in the system is adjusted to be in the above range. In addition, after the reaction rate becomes steady, it is possible to adjust the addition amount, for example, 0.7 to 1.5 times that of the saccharified substrate.

The aqueous medium added to the reaction system is added so that the substrate concentration of the reaction system is within the above range.
The amount of cellulase added to the reaction system is generally about 0.5 to 50% by weight, preferably about 0.5 to 30% by weight, based on the substrate (cellulose content) added at the start of the reaction. When the amount of cellulase added is less than 0.5% by weight, the enzyme reactivity is slow and the time required for the reaction becomes too long, which is not efficient. When the amount of cellulase added exceeds 50% by weight, the reactivity is Although it increases, there is a problem in cost effectiveness, which is not preferable.

  The reaction temperature and pH are within the range in which the cellulase used acts, and preferably the optimum conditions for each enzyme. When a commercially available general enzyme is used, the reaction temperature is preferably 20 to 60 ° C. and the pH is preferably 3.0 to 8.0, if exemplified. In addition, the reaction time can be appropriately determined depending on the type, concentration, addition amount, etc. of cellulase and substrate, and is adjusted at the addition rate when adding the substrate etc. continuously, and at the addition interval when adding sequentially. be able to.

  After starting the reaction, it is preferable to stir the reaction vessel so that the substrate is uniformly dispersed in the system, since the progress of the reaction can be accelerated. The stirring method may be performed by any method such as rotation, shaking, circulation by a pump, or blowing using a gas that does not affect the enzyme. In the case of rotation, depending on the shape and size of the blade, for example, 50 to 400 rpm is appropriate. Excessive stirring is not preferable because it leads to inactivation of the enzyme.

  The mode of addition of cellulose or cellulose-containing substance and aqueous medium into the reaction system can be either continuous addition or sequential addition. The rate of addition in the case of continuous addition, the interval between additions in the case of sequential addition, and the amount added once are the amount of substrate to be added, the initial amount of cellulase added, the concentration of substrate and cellulase in the reaction system, and other conditions. Can be appropriately set so as to fall within the above range. The addition interval can be, for example, 3 to 48 hours.

  In the present invention, as described above, cellulose or a cellulose-containing substance and an aqueous medium are added to the reaction system, while the reaction solution is withdrawn (removed) from the reaction system continuously or sequentially. Continuous means a form that is continuously extracted from the reaction system, while sequential means that it is extracted from the reaction system in a predetermined cycle. The amount (rate) with which the reaction solution is withdrawn from the reaction system is preferably such that the reaction solution in the reaction system is replaced in 3 to 48 hours, and need not be constant throughout the reaction. The amount of substrate to be added, the initial addition of cellulase It can be changed arbitrarily according to conditions such as amount. If the amount of the aqueous medium to be added and the amount of the reaction solution to be extracted is increased, the concentration of cellooligosaccharide is decreased, but the amount of glucose produced as a by-product is decreased, so that the yield rate of cellooligosaccharide relative to the substrate is improved.

When extracting the reaction liquid from the reaction system, a separation device is provided at the outlet from the reaction tank, and the solid content of cellulose (that is, cellulose or cellulose-containing substance) as a substrate and the reaction saccharified liquid containing cellooligosaccharide are extracted. Separation may be performed at the same time (FIG. 1), or after removing the reaction solution from the reaction tank, the cellulose solid and the reaction saccharified solution containing cellooligosaccharide may be separated (FIG. 2). It is preferable to carry out extraction and separation at the same time for continuous production.
When extracting and separating simultaneously, in the separation device, install a membrane, filter, filter cloth, etc. on the surface that comes into contact with the reaction solution from the reaction tank to separate the solid content of cellulose as a substrate and the reaction saccharified solution. Can do. Non-cellulose non-woven fabrics are less likely to cause clogging and are preferable from the viewpoint of reaction efficiency. Of course, screens, metal filters, ceramic filters, and the like can also be used. Further, only a clear reaction saccharified solution can be recovered by decantation.

  In the case of separation after extraction, the reaction solution is separated using a known apparatus or a membrane, filter, filter cloth or the like as described above. At this time, the separated cellulose solid content is preferably returned to the reaction system. As a result, raw materials can be saved and the amount of waste can be reduced, and the solid content is already in a state in which cellulase is adsorbed to cellulose in the reaction system, so that the influence of β-glucosidase is eliminated as described later. Can do. Further, by returning the separated cellulose solid content to the reaction system, it is possible to recover and reuse components effective for the production of cellooligosaccharide adsorbed on cellulose.

  In addition, as a known apparatus used for separation of the cellulose or the cellulose-containing substance, a disk filter, a drum filter, a filter press, a screw press, a (tilt) screen, a (continuous) centrifuge, a decanter, a (vacuum) belt filter, a thickener , Oliver filters, pre-coated filters, fluid cyclones, etc. can be used. In addition, the reaction liquid roughly separated by the known known apparatus can be further solid-liquid separated with a membrane, a filter, a filter cloth or the like, and a clear reaction saccharified liquid can be recovered by decantation.

  The reaction saccharified solution contains cellooligosaccharide and glucose, and the cellooligosaccharide can be separated from the reaction saccharified solution by a known method. For example, after concentrating the cellooligosaccharide content with a reverse osmosis membrane device, further concentrating with an evaporator or the like, and crystallization from the concentrated solution or in a hydrous solvent such as methanol, ethanol, propanol, isopropanol, acetone, etc. It is easy to collect by precipitation. Alternatively, glucose coexisting with the cellooligosaccharide can be separated from the solution after the reverse osmosis membrane treatment by an adsorption treatment with a chromatographic separation apparatus or activated carbon or the like. In addition, microorganisms that selectively assimilate only glucose, such as yeast, can assimilate glucose to separate cellooligosaccharides. Of course, depending on the application, cellooligosaccharide and glucose may be used without being separated. Note that the reaction saccharified solution may be used as a concentrated solution or may be separately subjected to a known purification step and / or drying step.

  As another feature of the present invention, in order to eliminate the influence of β-glucosidase contained in cellulase at the initial stage of reaction or in the middle of the reaction, cello-oligo among enzyme components contained in cellulase is used as a substrate to be added to the reaction system. Cellulose or a cellulose-containing substance in which only enzymes necessary for sugar production (enzymes other than β-glucosidase, mainly endoglucanase, cellobiohydrase, etc.) are adsorbed in advance to cellulose or a cellulose-containing substance can be used. Thereby, cellooligosaccharide can be manufactured with a high yield.

  As a method of preliminarily adsorbing enzyme components other than β-glucosidase among the enzyme components contained in cellulase to cellulose, cellulose or a cellulose-containing substance is suspended in an appropriate liquid (buffer solution, water, etc.) A cellulose slurry having a pH and a concentration is preferably added. Cellulase is added to the slurry, and the mixture is kept at a constant temperature and time, and then the solution is removed and washed with water.

  The pH of the cellulose slurry is pH 5.0 to 10.0, preferably pH 5.5 to 9.0, more preferably pH 6.0 to 8.0, and particularly preferably around pH 7.0. If the pH is less than 5.0, the affinity of β-glucosidase for cellulose is improved, and if the pH exceeds 10.0, cellulase may be deactivated, which is not preferable.

  The cellulose slurry concentration is 1 to 10% by weight, preferably 1 to 8% by weight, and more preferably 1 to 7% by weight. When the slurry concentration is less than 1% by weight, it becomes dilute, so the degree of contact between cellulase and cellulose is reduced, and when the slurry concentration exceeds 10% by weight, the fluidity of cellulose is remarkably reduced, and workability is improved. Since the amount of adsorption of enzyme components other than β-glucosidase deteriorates, it is not preferable.

  The amount of cellulase added to cellulose or a cellulose-containing substance is 5 to 300% by weight, preferably 7 to 200% by weight, more preferably 10 to 100% by weight, based on cellulose. When the amount of cellulase added is less than 5% by weight, the production rate of cellooligosaccharides during the enzyme reaction is slow, and when it exceeds 300% by weight, the amount of cellulase that cannot be adsorbed increases.

  The adsorption treatment temperature is usually 5 to 60 ° C, preferably 10 to 50 ° C, more preferably 20 to 40 ° C. When the temperature is lower than 5 ° C., the adsorption rate of cellulase decreases, and when the temperature exceeds 60 ° C., cellulase may be deactivated, which is not preferable.

  The adsorption treatment time is usually 1 to 150 minutes, preferably 1 to 100 minutes, and more preferably 1 to 60 minutes. When the adsorption treatment time is less than 1 minute, enzyme components other than β-glucosidase (mainly endoglucanase and cellobiohydrase) cannot be completely adsorbed. No change is observed in the amount of adsorbed enzyme components (mainly endoglucanase, cellobiohydrase), and further treatment leads to time loss, which is not preferable. Cellulase adsorption proceeds by natural diffusion without stirring, but is accelerated by stirring. The stirring is not particularly limited as long as the enzyme is not inactivated. If the cellulase is sufficiently agitated and evenly in contact with cellulose, the adsorption time will be short.

  The cellulose slurry after the adsorption treatment is subjected to liquid removal and water washing (washing) by a known solid-liquid separation method to eliminate enzyme components that are not adsorbed on cellulose, mainly β-glucosidase. In addition, in order to further remove β-glucosidase, washing and liquid removal may be repeatedly performed once or a plurality of times with 50 to 1000 times the amount of water of cellulose.

  The timing of adding cellulose or a cellulose-containing substance to which the enzyme component other than β-glucosidase among the enzyme components contained in cellulase obtained in this manner is preliminarily adsorbed is the initial stage of the reaction (at the same time as the start of the reaction or Immediately after), or during the reaction, or both the initial stage and the middle of the reaction. The middle of the reaction may be any time from the start of the reaction to the end of the reaction. The number of times of addition is not particularly limited, and when added during the reaction, it can be added twice or more.

  Among the enzyme components contained in the cellulase, cellulose that does not adsorb the enzyme is added as the addition amount in the case of adding cellulose or a cellulose-containing substance to which an enzyme component other than β-glucosidase is adsorbed in the initial stage and / or during the reaction. Including the case, the total amount of cellulase added to the reaction system can be adjusted to be in the above-described range with respect to the substrate (cellulose content) added at the start of the reaction. These can be added alone or in combination with cellulose not subjected to the above-described adsorption treatment or a cellulose-containing substance in an arbitrary ratio. As for the blending ratio, the amount of cellulase added (the amount of cellulase added during the enzyme adsorption treatment) with respect to the total cellulose content is 0.5 to 50% by weight, preferably 0.5 to 30% by weight. It is preferable to mix. If the amount of cellulase added to the total cellulose content (the amount of cellulase added during the enzyme adsorption process) is less than 0.5% by weight, the enzyme reactivity is slow and the time required for the reaction is too long, which is not efficient. When the amount of cellulase added exceeds 50% by weight, the reactivity increases, but there is a problem in cost effectiveness, which is not preferable.

[Action]
In the present invention, enzyme components (mainly endoglucanase and cellobiohydrase) contained in cellulases other than β-glucosidase are hardly found in the extracted reaction solution, and therefore, the enzyme is used in an expensive ultrafiltration membrane. There is no need to separate and recover only. This is because cellulase adsorbed on cellulose decomposes cellulose because the enzyme components that are largely involved in the production of cellooligosaccharides such as endoglucanase and cellobiohydrase have an adsorption site (cellulose binding domain) for cellulose. This is probably because most of the enzyme components remain in the reaction system in order to repeatedly bind to a new substrate (cellulose) added to the reaction system. In addition, it is considered that an enzyme adsorbed on cellulose or a cellulose-containing substance works as an immobilized enzyme in a part of the reaction system. On the other hand, β-glucosidase in the system theoretically has no adsorptivity to cellulose or cellulose-containing substances (has no cellulose binding domain), so it is discharged out of the system together with the extracted reaction solution, and the reaction is completed. Sometimes almost all of it is extracted out of the system.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to this.

<Quantitative determination of cellobiose and glucose>
Using high performance liquid chromatography, cellobiose and glucose concentrations were calculated from the peak area. The column was MCI GEL CK04S (manufactured by Mitsubishi Chemical Corporation), the eluent was water, the flow rate was 0.3 ml / min, and the temperature was 45 ° C., and a differential refractometer (manufactured by JASCO Corporation) was used. ).

[ Reference Example 1]
Dissolve the pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) in a 1 L Erlenmeyer flask containing 500 ml of 0.05 M acetic acid buffer (pH 5.0) so as to be about 5% by weight. 2 g of Celcrust (manufactured by Novozymes) is added and kept at 50 ° C. in a thermostatic bath. The Erlenmeyer flask is stirred (200 rpm) with a stirrer. Thereto, 2.5 g (dry weight) of the above-described dissolving pulp is added every 12 hours. While the same buffer solution is continuously added to the Erlenmeyer flask at a rate of 20 ml / hr, the reaction solution is continuously withdrawn at the same rate. The extraction port was covered with a non-cellulose non-woven fabric to prevent the pulp from being extracted from the reaction vessel. The reaction was continued for 10 days. The reaction liquids for 10 days were totaled, and when the cellooligosaccharide was quantified with a differential refractometer using a gel filtration column by a high performance liquid chromatograph according to a conventional method, about 38 g was contained. The cellooligosaccharide yield based on the added pulp was 50% by weight. By concentrating the extracted reaction solution, about 22 g of crystals having a cellobiose purity of 96% by volume were obtained.

  Table 1 shows the amount of cellooligosaccharide produced for each elapsed day. The extracted reaction solution contained an average of about 8 mg / ml of cellobiose and about 3 mg / ml of glucose in the reaction for 10 days.

  As is clear from Table 1, according to the present invention, it was found that a solution having a high cellooligosaccharide ratio can be easily and continuously collected.

[ Reference Example 2]
Dissolve the pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) in a 500 ml Erlenmeyer flask containing 300 ml of 0.05M acetic acid buffer (pH 5.0) so as to be about 5% by weight. 5 g of Celcrust (manufactured by Novozymes) is added and kept at 50 ° C. in a thermostatic bath. The Erlenmeyer flask is stirred (200 rpm) with a stirrer. After the reaction for 24 hours, the contents of the Erlenmeyer flask are collected by centrifugation (10,000 rpm). The recovered cellulose part was suspended again in a 500 ml Erlenmeyer flask containing 300 ml of 0.05 M concentration acetate buffer (pH 5.0), 5 g (dry weight) of the substrate was newly added thereto, and the temperature was adjusted to 50 ° C. in a thermostatic bath. Keep warm. The Erlenmeyer flask is stirred (200 rpm) with a stirrer.

  This operation is repeated for 7 days. When the reaction liquid collected for 7 days obtained by centrifugation was analyzed, it was found that about 23 g of cellooligosaccharide was contained. Glucose was about 7 g. The yield of cellooligosaccharide based on the pulp was about 51% by weight.

[ Reference Example 3]
Dissolving pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) in a 1 L Erlenmeyer flask containing 500 ml of 0.05M acetate buffer pH 5.0 so as to be about 5% by weight, and Celcrust (Novozymes) 2 g of Co., Ltd.) is added and stirred at 20 ° C. for 3 hours, and then the cellulose portion is recovered using a Kiriyama funnel. The recovered cellulose was washed with 100 ml of water. The cellulose adsorbed with cellulase is suspended in a 1 L Erlenmeyer flask containing 500 ml of the same buffer so as to be about 5% by weight, and kept at 50 ° C. The Erlenmeyer flask was stirred (200 rpm) with a stirrer. There, melt pulp (dry weight 2.5g) is added every 12 hours. While continuously adding a buffer solution (without dissolving pulp) to the Erlenmeyer flask at a rate of 20 ml / hr, the reaction solution is continuously withdrawn at the same rate. The extraction port is covered with a non-cellulose non-woven fabric so that pulp is not extracted from the reaction vessel. The reaction was continued for 10 days.

The reaction liquids for 10 days were totaled, and the cellooligosaccharide was quantified with a differential refractometer using a gel filtration column by a high performance liquid chromatograph according to a conventional method. The cellooligosaccharide yield based on the added pulp was 57% by weight.
By concentrating the extracted reaction solution, about 23 g of crystals having a cellobiose purity of 97% by volume were obtained.
Table 2 shows the amount of cellooligosaccharide produced for each elapsed day. The extracted reaction solution contained 9.0 mg / ml cellobiose and 2.9 mg / ml glucose on average for 10 days.

  From the results in Table 2, it was found that cellooligosaccharides can be produced more efficiently by using cellulose adsorbed with an enzyme component other than β-glucosidase of cellulase.

[ Reference Example 4]
500 ml of 0.05M acetic acid buffer solution (pH 5.0) charged with dissolving pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) so that the pulp concentration is 5% by weight is placed in a 1 L Erlenmeyer flask, Cellcrust (manufactured by Novozymes) was added at 10% by weight to the pulp and kept at 50 ° C. in a thermostatic bath. While stirring the pulp slurry with a stirrer (200 rpm), the above-described dissolving pulp slurry (slurry concentration 1.0 wt%) is continuously added to the above Erlenmeyer flask at a rate of 20 ml / hr, while the extraction port is not The reaction saccharified solution was continuously withdrawn at the same speed in a state where it was covered with a nonwoven fabric made of cellulose and the pulp was not leaked from the reaction vessel. The reaction was carried out continuously for 10 days, and cellobiose and glucose concentrations were measured every day according to a conventional method, and the production amount of each sugar was calculated from the extracted amount. In addition, the cellobiose yield with respect to the added pulp was about 52 weight%.

[Example 5]
100 ml of ion-exchanged water (pH 7.0) charged with dissolving pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) so that the pulp concentration is 5% by weight is put into a 500 ml Erlenmeyer flask, and Celcrust (Novozymes (Novozymes ( Co., Ltd.) was added to 50% by weight of pulp and stirred (200 rpm) at 20 ° C. for 10 minutes, and then the cellulose content was recovered using a Kiriyama funnel. The recovered cellulose was washed with 100 ml of water. The cellulose adsorbed with cellulase and 20 g of the above-mentioned dissolving pulp (dry weight) were put into a 1 L Erlenmeyer flask, and 0.05 M acetic acid buffer solution (pH 5.0) was added so as to be 5% by weight. Kept warm. While stirring the pulp slurry with a stirrer (200 rpm), 2.5 g (dry weight) of the above-described dissolving pulp was added every 12 hours. In addition, while the same buffer solution was continuously added to the aforementioned Erlenmeyer flask at a rate of 20 ml / hr, the extraction port was covered with a non-cellulose nonwoven fabric so that the pulp did not leak out from the reaction vessel. The reaction saccharified solution was continuously extracted at the same rate. The reaction was carried out continuously for 10 days, and cellobiose and glucose concentrations were measured every day according to a conventional method, and the production amount of each sugar was calculated from the extracted amount. The cellobiose yield based on the pulp was about 60% by weight.

[Reference Example 6]
500 ml of 0.05M acetic acid buffer solution (pH 5.0) charged with dissolving pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) so that the pulp concentration is 5% by weight is placed in a 1 L Erlenmeyer flask, Cellcrust (manufactured by Novozymes) 10% by weight of pulp was added and stirred at 200 ° C. for 150 minutes (200 rpm), and then the cellulose content was recovered using a Kiriyama funnel. The recovered cellulose was washed with 100 ml of water. The cellulose adsorbed with cellulase was put into a 1 L Erlenmeyer flask, and the same buffer was added so as to be 5% by weight, and kept at 50 ° C. While stirring the pulp slurry with a stirrer (200 rpm), 2.5 g (dry weight) of the above-described dissolving pulp was added every 12 hours. In addition, while the same buffer solution was continuously added to the aforementioned Erlenmeyer flask at a rate of 20 ml / hr, the extraction port was covered with a non-cellulose nonwoven fabric so that the pulp did not leak out from the reaction vessel. The reaction saccharified solution was continuously extracted at the same rate. The reaction was carried out continuously for 10 days, and cellobiose and glucose concentrations were measured every day according to a conventional method, and the production amount of each sugar was calculated from the extracted amount. The cellobiose yield based on the pulp was about 52% by weight.

[Example 7]
300 ml of a dissolved pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) slurry adjusted to a pulp concentration of 10% by weight is treated with a PFI mill (manufactured by Kumagaya Riki Kogyo Co., Ltd.) until it reaches 10,000 revolutions to produce fibrils. A pulp was obtained.
500 ml of ion-exchanged water (pH 7.0) charged with the above-mentioned fibrillated pulp is added to a 1 L Erlenmeyer flask so that the pulp concentration becomes 5% by weight, and Cell Crust (manufactured by Novozymes) is paired with the aqueous solution. 10% by weight of pulp was added and stirred (200 rpm) at 20 ° C. for 120 minutes, and then the cellulose content was recovered using a Kiriyama funnel. The recovered cellulose was washed with 100 ml of water. The cellulose adsorbed with cellulase was put into a 1 L Erlenmeyer flask, and an acetic acid buffer solution (pH 5.0) having a concentration of 0.05 M was added so as to be 5% by weight, and kept at 50 ° C. While stirring the pulp slurry with a stirrer (200 rpm), 2.5 g (dry weight) of the above fibrillated pulp was added every 12 hours. In addition, while the same buffer solution was continuously added to the aforementioned Erlenmeyer flask at a rate of 20 ml / hr, the extraction port was covered with a non-cellulose nonwoven fabric so that the pulp did not leak out from the reaction vessel. The reaction saccharified solution was continuously extracted at the same rate. The reaction was carried out continuously for 10 days, and cellobiose and glucose concentrations were measured every day according to a conventional method, and the production amount of each sugar was calculated from the extracted amount. The cellobiose yield based on the pulp was about 63% by weight.

[Example 8]
333 ml of water suspension prepared by placing ceramic balls with a diameter of 30 mm in a 1 L pot so that the filling rate is 65% by volume and further dissolving pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) to a pulp concentration of 6% by weight. And treated for 30 minutes with a vibration ball mill (manufactured by Chuo Kako) to obtain mechanochemically treated pulp.
500 ml of ion-exchanged water (pH 7.0) charged with the above mechanochemically treated pulp was added to a 1 L Erlenmeyer flask so that the pulp concentration would be 5% by weight, and Celcrust (manufactured by Novozymes) was added to the aqueous solution. 10% by weight of pulp was added and stirred (200 rpm) at 20 ° C. for 120 minutes, and then the cellulose content was recovered using a Kiriyama funnel. The recovered cellulose was washed with 100 ml of water. The cellulose adsorbed with cellulase was put into a 1 L Erlenmeyer flask, and an acetic acid buffer solution (pH 5.0) having a concentration of 0.05 M was added so as to be 5% by weight, and kept at 50 ° C. While stirring the pulp slurry with a stirrer (200 rpm), 2.5 g (dry weight) of the above mechanochemically treated pulp was added every 12 hours. In addition, while the same buffer solution was continuously added to the aforementioned Erlenmeyer flask at a rate of 20 ml / hr, the extraction port was covered with a non-cellulose nonwoven fabric so that the pulp did not leak out from the reaction vessel. The reaction saccharified solution was continuously extracted at the same rate. The reaction was carried out continuously for 10 days, and cellobiose and glucose concentrations were measured every day according to a conventional method, and the production amount of each sugar was calculated from the extracted amount. The cellobiose yield based on the pulp was about 60% by weight.

[Example 9]
Dissolving pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) was immersed in a 15 wt% aqueous sodium hydroxide solution adjusted to a pulp concentration of 5 wt%, stirred at room temperature for 30 minutes (200 rpm), and then centrifuged. After removing excess alkali, it was washed with ion-exchanged water until the pH became neutral to obtain an alkali-treated pulp.
500 ml of ion-exchanged water (pH 7.0) charged with the above-mentioned alkali-treated pulp is added to a 1 L Erlenmeyer flask so that the pulp concentration becomes 5% by weight, and Celcrust (manufactured by Novozymes) is added to the aqueous solution. 10% by weight of pulp was added and stirred (200 rpm) at 20 ° C. for 120 minutes, and then the cellulose content was recovered using a Kiriyama funnel. The recovered cellulose was washed with 100 ml of water. The cellulose to which cellulase was adsorbed was put into a 1 L Erlenmeyer flask, and an acetic acid buffer solution (pH 5.0) having a concentration of 0.05 M was added so as to be 5% by weight, and kept at 50 ° C. While stirring the pulp slurry with a stirrer (200 rpm), 2.5 g (dry weight) of the aforementioned alkali-treated pulp was added every 12 hours. In addition, while the same buffer solution was continuously added to the aforementioned Erlenmeyer flask at a rate of 20 ml / hr, the extraction port was covered with a non-cellulose nonwoven fabric so that the pulp did not leak out from the reaction vessel. The reaction saccharified solution was continuously extracted at the same rate. The reaction was carried out continuously for 10 days, and cellobiose and glucose concentrations were measured every day according to a conventional method, and the production amount of each sugar was calculated from the extracted amount. The cellobiose yield based on the pulp was about 62% by weight.

[Example 10]
Dissolving pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) was immersed in a 15 wt% aqueous sodium hydroxide solution adjusted to a pulp concentration of 5 wt%, stirred at room temperature for 30 minutes (200 rpm), and then centrifuged. After removing excess alkali, it was washed with ion-exchanged water until the pH became neutral to obtain an alkali-treated pulp. Using the alkali-treated pulp as a raw material, 300 ml of pulp slurry adjusted to a pulp concentration of 10% by weight is treated with a PFI mill (manufactured by Kumagaya Riki Kogyo Co., Ltd.) until it reaches 10,000 rotations, and alkali-treated fibrils are obtained. A pulp was obtained.
500 ml of ion-exchanged water (pH 7.0) charged with the above-mentioned alkali-treated fibrillated pulp was added to a 1 L Erlenmeyer flask so that the pulp concentration was 5% by weight, and Celcrust (manufactured by Novozymes) was added to the aqueous solution. Was added at 10% by weight to the pulp and stirred (200 rpm) at 20 ° C. for 120 minutes, and then the cellulose content was recovered using a Kiriyama funnel. The recovered cellulose was washed with 100 ml of water. The cellulose adsorbed with cellulase was put into a 1 L Erlenmeyer flask, and an acetic acid buffer solution (pH 5.0) having a concentration of 0.05 M was added so as to be 5% by weight, and kept at 50 ° C. While stirring the pulp slurry with a stirrer (200 rpm), 2.5 g (dry weight) of the aforementioned alkali-treated fibrillated pulp was added every 12 hours. In addition, while the same buffer solution was continuously added to the aforementioned Erlenmeyer flask at a rate of 20 ml / hr, the extraction port was covered with a non-cellulose nonwoven fabric so that the pulp did not leak out from the reaction vessel. The reaction saccharified solution was continuously extracted at the same rate. The reaction was carried out continuously for 10 days, and cellobiose and glucose concentrations were measured every day according to a conventional method, and the production amount of each sugar was calculated from the extracted amount. The cellobiose yield based on the pulp was about 64% by weight.

[Comparative Example 1]
500 ml of 0.05M acetic acid buffer solution (pH 5.0) charged with dissolving pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) so that the pulp concentration is 5% by weight is placed in a 1 L Erlenmeyer flask, Celcrust (manufactured by Novozymes) was added at 10% by weight to the pulp, and the mixture was kept at 50 ° C. in a thermostatic bath while stirring (200 rpm) with a stirrer. While the same buffer solution was continuously added to the aforementioned Erlenmeyer flask at a rate of 20 ml / hr, the extraction port was covered with a non-cellulose non-woven fabric so that the pulp did not leak from the reaction vessel. The reaction saccharified solution was continuously withdrawn. The reaction was carried out continuously for 10 days, and cellobiose and glucose concentrations were measured every day according to a conventional method, and the production amount of each sugar was calculated from the extracted amount. In addition, the cellobiose yield with respect to the added pulp was about 49 weight%.

[Comparative Example 2]
500 ml of 0.05M acetic acid buffer solution (pH 5.0) charged with dissolving pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) so that the pulp concentration is 5% by weight is placed in a 1 L Erlenmeyer flask, Cell crust (manufactured by Novozymes) 10% by weight of pulp was added, and the reaction was continued for 10 days while being kept at 50 ° C. in a thermostatic bath while being stirred (200 rpm) with a stirrer. According to a conventional method, cellobiose and glucose concentrations were measured every day, and the production amount of each sugar was calculated from the internal volume. The cellobiose yield based on the pulp was about 2% by weight.

[Comparative Example 3]
500 ml of 0.05M acetic acid buffer (pH 5.0) charged with dissolving pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) so that the pulp concentration is 5% by weight is placed in a 1 L Erlenmeyer flask, and the aqueous solution is added to the cell crust. (Novozymes Co., Ltd.) 10% by weight with respect to pulp was added, stirred at 200 ° C. for 120 minutes (200 rpm), and then the cellulose content was recovered using a Kiriyama funnel. The cellulose adsorbed with cellulase was put into a 1 L Erlenmeyer flask, and the same buffer was added so as to be 5% by weight, and kept at 50 ° C. The reaction was continued for 10 days while stirring the pulp slurry with a stirrer (200 rpm). According to a conventional method, cellobiose and glucose concentrations were measured every day, and the production amount of each sugar was calculated from the internal volume. The cellobiose yield based on the pulp was about 3% by weight.

[Comparative Example 4]
Cell crust (manufactured by Novozymes) was added to 500 ml of 0.05M acetic acid buffer (pH 5.0) charged with dissolving pulp (manufactured by Nippon Paper Chemicals Co., Ltd.) so that the pulp concentration was 5% by weight. Was added to an ultrafilter with a stirrer (flat membrane type) in which a polysulfone flat membrane with a molecular weight cut off of 10,000 was set so that β-glucosidase remained in the reaction system. The temperature of the stirrer was kept at 200 ° C. and 50 ° C. While adding 2.5 g (dry weight) of the above-described dissolving pulp every 12 hours, the buffer solution is continuously added at a rate of 20 ml / hr, while the ultrafilter is 0.1 to 0.3 MPa. The reaction saccharified solution was continuously withdrawn at the same speed by adjusting the pressure. The reaction was carried out continuously for 10 days, and cellobiose and glucose concentrations were measured every day according to a conventional method, and the production amount of each sugar was calculated from the extracted amount. In addition, the cellobiose yield with respect to the added pulp was about 42 weight%.

  Table 3 shows the test results of Examples 4 to 10 and the comparative example, and Table 4 shows time-dependent changes in the cellulose concentration in the reaction systems of the examples and comparative examples.

As is apparent from Table 3, according to the present invention, it was found that a reactive saccharified solution having a high cellobiose concentration can be collected easily and continuously. Moreover, it discovered that cellooligosaccharide (cellobiose) could be manufactured more efficiently by using the cellulose which adsorb | sucked previously enzyme components other than (beta) -glucosidase contained in a cellulase. From the results of Examples 7 to 10, when cellulose used as a substrate was previously subjected to fibrillation treatment, alkali treatment, and mechanochemical treatment, the reactivity of cellulose was improved, and the cellobiose concentration was increased from the initial stage of the reaction. It turned out that the reaction efficiency increased.
From Table 4, as in Comparative Examples 1 to 4, when the substrate is not added during the reaction or when β-glucosidase is not discharged from the system, the cellulose concentration cannot be maintained at 1% by weight or more. The cellooligosaccharide production rate decreased, whereas in Examples 1 to 7, it was revealed that the cellulose concentration could be maintained even 10 days after the start of the reaction.

It is a figure which shows the outline of the reaction system of the main aspect of this invention. It is a figure which shows the outline of the reaction system of the main aspect of this invention.

Claims (3)

In a reaction system to produce the cellooligosaccharide reacting the cellulase to cellulose or cellulose-containing materials, while successively adding the sequential addition to an aqueous medium the cellulose or cellulose-containing materials in the reaction system, continuously outside of the reaction system in the reaction solution Extracting with a non-cellulose non-woven fabric, the enzyme component other than β-glucosidase among the enzyme components contained in cellulase for the cellulose or cellulose-containing substance added to the reaction system at the initial stage and / or during the reaction A method for producing a cellooligosaccharide characterized by pre-adsorbing and maintaining the cellulose concentration in the reaction system at 1% by weight or more ,
Cello-oligo treated by adding 5 to 300% by weight of cellulase to cellulose slurry in which cellulose is dissolved in water to adjust the pH to 7.0 and the slurry concentration is adjusted to 1 to 10% by weight. A method for producing sugar .   The reaction liquid extracted outside the reaction system is separated into a reaction saccharification liquid and cellulose or a cellulose-containing substance, and then the separated cellulose or cellulose-containing substance is returned to the reaction system. A method for producing cellooligosaccharides. The cellooligosaccharide production according to claim 1 or 2 , wherein the cellulose or the cellulose-containing substance is a pulp subjected to at least one of fibrillation treatment, mechanochemical treatment, and chemical treatment. Method.

Images