JP2006034206A - METHOD FOR PRODUCING CELLOBIOSE, METHOD FOR SEPARATION AND REMOVAL OF beta-GLUCOSIDASE AND METHOD FOR RECOVERY OF CELLULASE - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for producing cellobiose having improved purity at a low cost by using a simplified production process compared with conventional cellobiose production methods and a method for separating and removing β-glucosidase from cellulase to eliminate the β-glucosidase active to excessively decomposing the produced cellobiose. <P>SOLUTION: The method for producing cellobiose comprises the charge of a cellulosic raw material 3 in a tubular or column reactor 1, the continuous introduction of a cellulase solution containing a cellobiohydrolase in one direction to effect the contact and reaction of the cellulosic raw material with the cellulase solution and the separation of a fraction containing the cellobiose from the discharged solution. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing cellobiose used in sweeteners, pharmaceuticals, health foods, dietary fibers, reagents, and the like, and a method for separating and removing β-glucosidase from cellulase containing cellobiohydrolase important for cellobiose production. It is.

  Cellobiose is a useful saccharide used in sweeteners, pharmaceuticals, health foods, dietary fiber, reagents and the like.

  Until now, cellobiose has been produced by an acid hydrolysis reaction of cellulosic raw materials or an enzymatic hydrolysis reaction with cellulase. In the acid hydrolysis reaction, the cellobiose yield was low, and it was necessary to purify cellobiose from a large amount of by-products. In addition, the acid hydrolysis method is not an industrially simple method for producing cellobiose such as requiring high pressure and acid resistant equipment.

  On the other hand, in the enzymatic hydrolysis reaction, the reaction conditions are mild, and no high pressure, acid resistant equipment, etc. are required. However, since cellulase is a complex enzyme system containing cellobiohydrolase, β-glucosidase, endoglucanase and the like, there is a disadvantage that glucose is mainly produced and the ratio of cellobiose is lowered.

Thus, several techniques have been proposed for the production of cellobiose. Patent Document 1 proposes a production method that does not use a cellulosic material. Patent Document 2 proposes a method of obtaining cellobiose using an enzyme from which β-glucosidase in cellulase is removed using chitosan and β-glucosidase is removed. This method cannot be said to be a simple method because the process of separating and removing β-glucosidase is complicated. Patent Document 3 is a technique that enables cellulosic raw materials containing lignin to be used by allowing lignin-degrading bacteria to coexist without subjecting to delignification treatment. Filtration equipment is used. In these apparatuses, the content of cellobiose in the reaction product is low, and purification of cellobiose is necessary. Patent Document 4 proposes a method of (1) selecting a raw material containing a large amount of amorphous cellulose in order to increase the cellobiose content, and (2) performing an enzyme reaction in the presence of lignin. . However, a specific method for collecting cellobiose as needed is to perform an enzymatic reaction in a tank reactor and collect the produced cellobiose using an ultrafiltration device. Therefore, an ultrafiltration device is an essential technique as a device. In Patent Document 5, the yield of cellobiose is increased by using bleached slush pulp and sulfite pulp having a specific water retention and freeness as raw materials, but the material characteristics are not mentioned in the enzyme reactor. It is a technology based on Patent Document 6 is characterized in that the raw material containing natural lignocellulose is digested to increase the decomposition efficiency by using an undried raw material, and the sugar solution is recovered using an ultrafiltration membrane with a molecular weight cut off of 10,000. Yes. Again, in order to increase the cellobiose content, an ultrafiltration device is an essential technique as a device. In Patent Document 7, it is proposed to selectively remove β-glucosidase in cellulase using cellulose ester or cellulose ether ester, and to carry out an enzyme reaction, but a separation step during enzyme purification is essential. Solubilization and insolubilization are performed before and after the reaction, and the process is complicated. Patent Document 8 utilizes the knowledge that β-glucosidase has a weak adsorption power to cellulose compared to other cellulase components, adsorbs cellulase components other than β-glucosidase to insoluble cellulose, and separates β-glucosidase by separation filtration. After selective removal, the solid fraction is suspended in an aqueous medium using an aqueous medium to carry out the enzyme reaction. The β-glucosidase removal step and the decomposition reaction step were separated. Furthermore, a large amount of washing liquid was required for separation and filtration of the solid fraction.
JP 2001-112896 A JP-A-5-227958 JP-A-8-89274 JP-A-5-317073 JP-A-9-107987 Japanese Patent Laid-Open No. 7-184678 JP-A-5-227957 Patent No. 1948184

An object of the present invention, as compared with the conventional cellobiose production method can simplify the manufacturing process, decomposition cellobiose is a further method of producing cellobiose can be obtained at low cost with high cellobiose purity, and product It is another object of the present invention to provide a method for separating and removing β-glucosidase which separates β-glucosidase from cellulase simultaneously with the production of cellobiose.

As a result of studying the action mechanism of cellulase on cellulosic substances with the aim of solving the above problems, the present inventor filled a cellulosic raw material into a reactor capable of solid-liquid separation, and passed the enzyme liquid as a mobile phase. When cellulase is allowed to act while liquid, the cellulosic raw material is efficiently decomposed, and the cellobiose concentration in the product can be significantly improved as compared with the tank reactor, through this reaction, the cellobiose is a product decomposed to cause β- glucosidase found that can be separated from the cellulase, thereby completing the present invention.

  That is, the present invention has the following main points.

  (1) A cellulosic raw material is charged into a tubular or tower reactor, a cellulase solution containing cellobiohydrolase is continuously passed from one direction, and the cellulosic raw material and the cellulase solution are brought into contact with each other to react. A method for producing cellobiose, comprising obtaining a fraction containing cellobiose from a flowing solution.

  (2) Before passing the cellulase solution or during the entire period of the reaction, only the gap between the cellulosic raw materials charged in the reactor is replaced with the liquid phase, and the excess liquid phase is eliminated. The method for producing cellobiose according to (1).

  (3) A cellulosic raw material is charged into a tube-type or tower-type reactor, and a cellulase solution containing cellobiohydrolase and containing β-glucosidase as an impurity is continuously passed from one direction to contain β-glucosidase. A method for separating and removing β-glucosidase from cellulase, wherein the fraction is selectively separated and removed.

  (4) The method for producing cellobiose according to (1), wherein the reaction solution from which β-glucosidase has been removed by the above method is repeatedly passed through a tubular or tower reactor.

(5) beta-grayed Le Koshidaze a mixture of cellulase solution and cellobiose containing cellobiohydrolases that has been removed, was passed through a tubular cellulosic feedstock has been filled or tower reactor, only the enzyme component A method for recovering cellulase, comprising adsorbing to a reactor and eluting only cellobiose.

  According to the method for producing cellobiose of the present invention, high-purity cellobiose can be obtained at low cost by a simple production process.

  Moreover, according to the method for separating and removing β-glucosidase of the present invention, β-glucosidase can be efficiently separated and removed simultaneously with the production of cellobiose.

  The cellulosic raw material used in the present invention is not limited as long as it contains cellulose in a form in which cellulase can act, such as waste paper, pulp, and woody material obtained by decomposing lignin. However, in order to efficiently perform the enzyme reaction, it is preferable to make it finer so as to become a powder or slurry.

  The cellulase used in the present invention is not particularly limited as long as it is an enzyme containing cellobiohydrolase. The cellulase obtained from can be illustrated. In the present invention, cellulases having close to optimum pH can be mixed and used. In addition, a cellulase-unpurified culture of the above-mentioned bacteria can be used as long as it has cellulase activity. Cellulase contains β-glucosidase that degrades cellobiose, which is a product, as an impurity. However, β-glucosidase can be efficiently separated and removed by the method of the present invention described in claim 3. Can do.

  The reactor used in the present invention is generally called a tubular or tower reactor (hereinafter sometimes abbreviated as “reactor”). In the reactor of the present invention, a cellulosic raw material, which is a solid raw material, is filled in a tubular or tower reactor, and a cellulase solution containing cellobiohydrolase (hereinafter sometimes abbreviated as “enzyme solution”) is used. The solid material of the cellulosic material is prevented from leaking. That is, a filter having a pore size that does not allow cellulosic materials to pass through may be provided in the reactor so that the cellulosic materials do not leak.

  In order to allow the enzyme solution to flow through the reactor filled with the cellulosic raw material in one direction, it may be a downward flow or an upward flow. When the enzyme solution is allowed to flow in a downward flow, a filter may be provided at both the upper and lower ends of the reactor, or a filter may be provided only at the lower end of the reactor. When the enzyme solution is allowed to flow in an upward flow, it is possible to prevent the cellulosic raw material from leaking by providing filters at the upper and lower ends of the reactor. In addition, there exists an advantage that an air layer is hard to generate | occur | produce in the clearance gap between a cellulosic raw material when the enzyme liquid is made to flow by an upward flow from the bottom to the top.

  Examples of the pore size filter through which the cellulosic material does not pass include a filter having a pore size of 0.1 to 3000 micrometers, and the material thereof is not particularly limited as long as it is resistant to cellulase. The relationship between the cross-sectional area and the length of the reactor is related to the speed of the reaction and can be changed as necessary. However, the length of the reactor is preferably 5 times or more the diameter of the cross-section.

  The tubular or tower reactor may be either vertical or horizontal.

  In the present invention, it is important to adjust the amount of the aqueous medium when filling the cellulosic material. That is, it is necessary to replace the gap between the cellulosic raw materials filled in the reactor with the liquid phase. Specifically, when a cellulosic raw material is charged into a tubular or tower reactor, an aqueous medium is added to sufficiently fill the solid content gap of the cellulosic raw material, and there is an excess aqueous medium. It is necessary not to create a space in the gap between the cellulosic raw material solids. Therefore, for example, a method in which the cellulosic raw material is first made into a slurry form, mixed in a state in which an excess aqueous medium is present, filled in a tubular or tower reactor, and the excess aqueous medium is allowed to flow out. .

  Also, when the cellulosic raw material decreases with the reaction, it is better to remove the excess aqueous medium.

  Cellulase containing cellobiohydrolase reacts with cellulosic material by flowing the enzyme solution from one direction into the reactor filled with cellulosic material, and the fraction containing cellobiose flows out from the reactor. . Cellobiose can be efficiently produced by collecting a fraction having a high cellobiose concentration in the reaction solution flowing out. The obtained cellobiose fraction can be purified by a known method to increase the purity of cellobiose.

  1 and 2 are flowcharts showing one embodiment of the present invention.

  As shown in FIG. 1, the reactor 1 of the present invention is a tubular or column type reactor, and the cellulosic raw material 3 packed in the reactor 1 is tubed or towered so as not to leak from the reactor. A filter 2 having a pore size through which cellulosic raw materials do not pass is provided at the end of the reactor.

  The cellulosic material and the enzyme solution are brought into contact with each other by causing the cellulase enzyme solution containing cellobiohydrolase to flow through the tube-type or tower reactor 1 filled with the cellulosic material 3 in one direction. The reaction liquid flows out from the tubular or tower reactor 1.

  In order to carry out the present invention, a flow path for filling the gap between the cellulosic raw materials 3 with an aqueous medium may be provided, and the aqueous medium may be passed by switching the valve 5. Further, a pump 4 for supplying an enzyme solution or an aqueous medium may be provided in the tower-type or tube-type reactor 1.

  As another embodiment, as shown in FIG. 2, the reactor 1 can be connected in series, and the substantial length of the reactor 1 can be increased. Furthermore, a discharge port may be provided in the middle of the reactor 1, and the discharge of the β-glucosidase fraction that flows out first may be removed early.

  The reactor preferably has a temperature holding mechanism such as a heater, but the temperature of the liquid phase to be passed may be held using a heat exchanger or the like.

  Examples of the medium used as the liquid phase in the present invention include an aqueous medium. Specifically, water, a salt solution, and the like can be used. However, the aqueous medium is preferably a buffer solution that can maintain the optimum pH (pH 3 to 9) of the cellulase used.

  The inside of the reactor is adjusted to a temperature at which sufficient enzyme activity is obtained, for example, 10 to 70 ° C.

  An aqueous medium as a mobile phase is passed from one side to the cellulosic raw material filled and fixed in such a state. When the reaction vessel is used in a vertical type, the liquid flow direction may be a downward flow or an upward flow, but at this time, the amount of the aqueous medium is maintained as described above with respect to the amount of the cellulosic raw material solids. It is necessary to adjust the amount of liquid flow and the amount of waste liquid. The liquid flow rate at this time is 0.1 to 35 cm / h, preferably 0.5 to 20 cm / h, as a linear flow rate with respect to the tubular or column reactor. This liquid flow starts immediately after the start of enzyme addition.

  The enzyme is first made into a solution and then added while passing through the mobile phase at the above flow rate. The amount of the enzyme solution is less than 10%, preferably less than 5% of the raw material filling volume.

  While the enzyme contacts and moves with the cellulosic raw material packed in the reactor, it repeatedly adsorbs and desorbs the cellulosic raw material component and decomposes the cellulosic raw material component, but β-glucosidase in the enzyme is It elutes first because of its weak adsorptive properties. In the meantime, cellulase decomposes cellulosic materials to produce cellobiose. Since cellobiose does not adsorb to the raw material, it is eluted sequentially after the elution of β-glucosidase. Therefore, the β-glucosidase elution fraction and the cellobiose fraction are collected separately.

  Cellulase in the reactor is eluted while continuing the decomposition reaction. This can be passed through a new reactor and continuously decomposed. It can also be returned to the same reactor. Since β-glucosidase has been removed once, the production of glucose is suppressed.

  The important point of the present invention is to keep the enzyme solution flowing at all times. By making it flow, cellulase reaction inhibition by cellobiose can be prevented, the enzyme reaction efficiency can be increased, and the amount of enzyme used can be greatly reduced compared to conventional tank-type reactions. .

  By separating and removing the fraction containing β-glucosidase from the first flowing solution, the cellobiose fraction obtained is mixed with cellulase from which β-glucosidase has been removed. Therefore, in order to reuse this, cellulase purified by ultrafiltration or the like can be concentrated and recovered, but the obtained cellobiose fraction is passed through a reactor filled with cellulosic raw materials. It is preferable to adsorb only cellulase. Moreover, the cellobiose fraction obtained after separating and removing β-glucosidase is again passed through a reactor filled with a cellulosic raw material, and by repeating this, a fraction rich in cellobiose can be obtained.

EXAMPLES Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited thereto.

  Cellulose powder Avicel (trade name, manufactured by Asahi Kasei Co., Ltd.) 5 g was suspended in 50 mM sodium acetate buffer (pH 5), and then packed into a chromatography glass column having a tube diameter of 16 mm and a length of 10 cm. Was used to remove excess aqueous medium. A mesh filter with a pore size of 10 micrometers was used at the outlet and inlet of the column to prevent Avicel from flowing out. The temperature was adjusted to 50 ° C., and 50 mM sodium acetate buffer (pH 5) was passed at a linear velocity of 5 cm / h. As for the enzyme, 0.02 g of commercially available cellulase preparation derived from Trichoderma genus, Mecelase (Meiji Seika) was dissolved in 1 ml of 50 mM sodium acetate buffer (pH 5), added from an injection valve, and then 50 mM acetic acid at a linear speed of 5 cm / h Sodium buffer (pH 5) was passed through. Five hours after the start of the reaction was separated as a fraction eluted with β-glucosidase, and the subsequent cellobiose fraction was repeatedly returned and allowed to react for 100 hours.

  The sugar composition of the eluted liquid was determined from the peak area using high performance liquid chromatography. The column used was KS-802 manufactured by Showa Denko, the eluent was water, and the detection was performed by differential refraction.

  As a control, 5 g of Avicel was suspended in 100 ml of 50 mM sodium acetate buffer (pH 5) in a beaker, and 0.02 g of mecerase was added and reacted at 50 ° C. for 100 hours.

  The results are shown in the table below.

  As described above, almost no cellobiose was obtained in the tank reactor, whereas the cellobiose content was increased and the yield was improved by using the tubular reactor of the present invention.

  The birch material was cooked at 185 ° C. for 15 minutes, and the material that could be decomposed by cellulase was made into a powder having a particle diameter of 1 mm. 700 g of this raw material was suspended in 50 mM sodium acetate buffer (pH 5), and then packed in a chromatography glass column having a tube diameter of 50 mm and a length of 100 cm, and an excess aqueous medium was removed using an adapter. A mesh filter having a pore diameter of 50 micrometers was used at the outlet and inlet of the column to prevent the wood flour from flowing out, and two of these were prepared. The temperature was adjusted to 50 ° C., and 50 mM sodium acetate buffer (pH 5) was passed at a linear velocity of 15 cm / h. As for the enzyme, 1 g of commercially available cellulase preparation derived from Trichoderma genus, Mecellase (trade name, manufactured by Meiji Seika Co., Ltd.) is dissolved in 15 ml of 50 mM sodium acetate buffer (pH 5), and added from the injection column through the first column inlet. Thereafter, for 10 hours, 50 mM sodium acetate buffer (pH 5) was passed at a linear velocity of 15 cm / h. For 10 hours from the start of the reaction, it was eluted from the outlet of the first column as a β-glucosidase elution fraction. Thereafter, two columns were connected in series, and the flow rate of the eluate was set to a linear velocity of 2 cm / h and reacted for 100 hours. The eluted cellobiose fraction was recovered as it was.

  The sugar composition of the eluted liquid was analyzed by the above method. The results are shown in Table 2.

  As described above, a liquid having a high cellobiose content could be recovered by the method of the present invention. Moreover, cellulase can be efficiently purified by removing the β-glucosidase elution fraction that elutes first.

  The cellobiose production method of the present invention is useful because the cellobiose having high purity can be obtained at low cost by a simple production process.

  Moreover, since the cellulase with low content of β-glucosidase can be obtained by the method for separating and removing β-glucosidase of the present invention, the present invention is useful.

The flowchart which shows one Embodiment of this invention. The flowchart which shows one Embodiment of this invention.

Explanation of symbols

1 Reactor 2 Filter 3 Cellulose Raw Material 4 Pump 5 Valve 6 Cellulase Return Pipe

Claims (5)

A cellulosic raw material is packed into a tube-type or tower-type reactor, a cellulase solution containing cellobiohydrolase is continuously passed from one direction, and the cellulosic raw material and the cellulase solution are brought into contact with each other to react and flow out. A method for producing cellobiose comprising obtaining a fraction containing cellobiose from Before the cellulase solution is allowed to flow or during the entire period of the reaction, only the gap between the cellulosic raw materials filled in the reactor is replaced with the liquid phase, and the excess liquid phase is eliminated. The method for producing cellobiose according to 1. A cellulosic raw material is charged into a tube-type or tower-type reactor, and a cellulase solution containing cellobiohydrolase and containing β-glucosidase as an impurity is continuously passed from one direction to obtain a fraction containing β-glucosidase. A method for separating and removing β-glucosidase from cellulase, which comprises selectively separating and removing. The method for producing cellobiose according to claim 1, wherein the reaction solution from which β-glucosidase has been removed by the above method is repeatedly passed through a tube-type or tower-type reactor. a mixture of cellulase solution and cellobiose containing cellobiohydrolases of β- grayed Le Koshidaze has been removed, was passed through a tubular cellulosic feedstock has been filled or tower reactor, only enzyme component to the reactor A method for recovering cellulase, which comprises adsorbing and eluting only cellobiose.

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