JP5038181B2 - Method for producing cellulase and cellooligosaccharide - Google Patents

Description

  The present invention relates to a method for producing cellulase in which β-glucosidase is selectively suppressed for efficiently obtaining cellooligosaccharide, and a method for producing cellooligosaccharide.

  Cellooligosaccharide is a generic name for cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose, and is a oligosaccharide having 1 to 6 glucopyranose units and β-1,4 linked. In recent years, cellooligosaccharides, like other oligosaccharides, are becoming clearer in physiological function and are expected as new materials for functional foods (see Non-Patent Document 1).

  In general, as a method for obtaining cellooligosaccharide, a method of decomposing a cellulosic substance with cellulase is known. The cellulose degradation mechanism by cellulase is as follows: (1) First, the reaction of cellulose degrading enzyme to produce cellooligosaccharides containing cellobiose from cellulose, (2) Reaction to produce glucose directly from cellulose, (3) β-glucosidase The reaction of producing glucose from cellooligosaccharides by this action is included (Non-patent Document 2). In this reaction system, in order to increase the productivity of cellooligosaccharide, it is effective to suppress β-glucosidase contained in cellulase, suppress the decomposition of cellooligosaccharide to glucose, and increase the selectivity of cellooligosaccharide.

  Conventionally, many studies have been made to increase the productivity of cellooligosaccharides by increasing the selectivity of cellooligosaccharides (Patent Documents 1 to 7). The difference between the existing technology and the present invention will be described below. Examples of methods for improving the selectivity of cellooligosaccharides by using specific cellulases and enzymatically decomposing cellulose include the following.

  Patent Document 1 discloses a method for producing cellooligosaccharide from a cellulosic substance in an aqueous reaction solution by the action of cellulase produced by a microorganism belonging to the genus Cellobibrio, which eliminates product inhibition by combining an ultrafiltration reactor. Thus, a method for producing cellooligosaccharides for producing and accumulating cellooligosaccharides is disclosed. If this method is used, an enzyme and a product can be isolate | separated by ultrafiltration and the activity fall of an enzyme can be prevented. However, the cellulase obtained by this method has a problem that it has a small effect on crystalline cellulose. Moreover, in order to obtain amorphous cellulose, since it was necessary to pre-process a cellulose-type substance, there existed a problem which a manufacturing process became complicated.

  In Patent Document 2, in a method of decomposing cellulose with cellulase to produce cellooligosaccharide, cellulase is contacted with a weakly acidic cation exchange resin previously equilibrated to pH 3.5 to 5.0, so that A method for producing cellooligosaccharides by selectively removing β-glucosidase and contacting cellulase from which β-glucosidase has been removed with cellulose is disclosed. According to this production method, glucose can be reduced by enzymatic degradation of cellulose, and a degradation product having 60% or more cellooligosaccharide can be obtained. However, the production method requires a step of removing β-glucosidase in cellulose, and there is a problem that the cellooligosaccharide production step becomes complicated. In addition, since this cellulase purification step requires a cation exchange resin 75 to 1000 times that of untreated cellulase, the amount of cellulase treatment is limited, cellooligosaccharide productivity is not sufficient, cellulase purification cost, There was a problem that the cost of the separation and purification agent for the cation exchange resin increased.

  Patent Document 3 discloses a method for obtaining cellobiose by insolubilizing cellulose or a cellulose-containing substance and trichoderma-derived cellulase in an aqueous medium, separating a solid fraction, and adding an aqueous solution to the solid fraction. Has been. Patent Document 4 describes a method for producing a cellooligosaccharide in which an enzyme component other than β-glucosidase is adsorbed in advance to cellulose or a cellulose-containing substance, and then enzymatically decomposed. In Patent Document 5, a cellulosic raw material is charged into a tube-type or tower-type reactor, a cellulase solution containing cellobiohydrolase and containing β-glucosidase as an impurity is continuously passed from one direction, and β- A method for separating and removing β-glucosidase from cellulase, which selectively separates and removes a fraction containing glucosidase, is described. In these production methods, β-glucosidase mixed in the cellobiose production process can be reduced, and cellobiose can be selectively obtained. However, these methods have a problem that the process is complicated because a cellulase adsorption step and a solid fraction separation step are required for the solid fraction.

  In contrast to the above-mentioned Patent Documents 2 to 5, the present invention provides β-glucosidase as it is from a cellulase suspension or an aqueous solution without passing through complicated steps such as treatment with an ion exchange resin and fractionation with cellulose. It is excellent in that it can be suppressed.

  Patent Document 6 discloses that the ratio of β-glucosidase activity to crystalline cellulose degrading activity (β-glucosidase activity / crystalline cellulose degrading activity) in a culture solution obtained by culturing cellulase-producing bacteria is 0.35 or less. A cellulase production method and a cellulase production method for controlling the pH in a culture solution to less than pH 3.5 are disclosed. Patent Document 7 discloses a method for producing an enzyme solution containing cellulase produced by inoculating a microorganism belonging to the genus Trichoderma into a liquid medium containing 0.1-10% by mass of cottonseed meal and culturing the microorganism. And the manufacturing method of the enzyme liquid in which a liquid culture medium contains the cellulase containing 0.01-10 mass% ammonium sulfate is indicated. Certainly, in these methods, an enzyme having high cellulolytic activity and low β-glucosidase can be obtained by highly controlling the culture conditions. On the other hand, according to the present invention, cellulase obtained by normal culture or commercially available cellulase can be treated without special culture, and Patent Document 6 and Patent Document 7 are completely different in this respect.

  As described above, conventionally, in order to efficiently obtain cellooligosaccharides, β-glucosidase activity in which cellulase is suspended or dissolved in an aqueous medium, brought to a specific pH of the suspension or aqueous solution, and heated to a specific temperature. There has been no known cellulase production method wherein cellulose is selectively suppressed and cellooligosaccharide production method using the cellulase.

Cellulose Communications, 5, No 2, 91-97 (1998) "Cellulase" Kodansha Scientific issue, 97-104 (1987) Japanese Patent Laid-Open No. 01-256394 JP 05-115293 A JP 63-226294 A JP 2006-204294 A JP 2006-34206 A JP 2006-296358 A JP 2007-215505 A

  The present invention relates to a cellulase production method capable of increasing the cellooligosaccharide selectivity in the selective production of cellooligosaccharide by enzymatic decomposition in the presence of cellulase using a cellulosic material, and the use of this cellulase. It is a problem to be solved to provide a method for producing cellooligosaccharides.

The present inventors have found that β-glucosidase activity can be selectively suppressed by heating cellulase at a specific pH and a specific temperature in the production of cellooligosaccharides that enzymatically degrade cellulosic substances. It came to achieve.
That is, the present invention is as follows.

(1) A method for producing cellulase with suppressed β-glucosidase activity, comprising suspending or dissolving cellulase in an aqueous medium, heating the suspension or aqueous solution to 7 or higher and heating to 30 ° C or higher.
(2) The method for producing cellulase with suppressed β-glucosidase activity according to claim 1, wherein the suspension or aqueous solution is heated to 30 ° C or higher in the presence of 0.01% by mass or more of a cellulose-based substance.
(3) (i) a step of producing cellulase in which β-glucosidase activity is suppressed by the method according to (1) or (2), and (ii) cellulose using the cellulase obtained in the step (i). A method for producing a cellooligosaccharide, comprising a step of decomposing a system substance.

  According to the present invention, a cellulase having high cellooligosaccharide selectivity can be produced, and cellooligosaccharide can be produced efficiently by using the cellulase for the production of cellooligosaccharide.

The cellulase of the present invention is derived from a culture solution obtained by culturing cellulase-producing bacteria.
The cellulase-producing bacterium used in the present invention is not particularly limited as long as it secretes an enzyme and the enzyme decomposes a cellulosic substance. Cellulase originates from, for example, the following microorganisms that produce known cellulases. Trichoderma (Tric h oderma) genus, Acremonium (Acremonium) spp, Aspergillus (Aspergillus) genus Bacillus (Bacillus) genus Pseudomonas (Pse-udomonas) genus Penicillium (Penicillium) genus, Aeromonas (Aeromonus) genus, Irupekkusu (Irpex ), Celluloses of the genus Sporotricum and the genus Humicola (published by Kodansha Scientific (1987)), and the bacteria described in “Encyclopedia of Cellulose” (published by Asakura Shoten (2000)) Cellulases that can be used are not limited to the enzymes derived from the above-mentioned known microorganisms, but also enzymes derived from novel bacteria may be used as long as they decompose cellulose. It is included in the hydrolase.

Particularly suitable among the above is a microorganism belonging to Tric h oderma genus. That is, inoculated with a microorganism belonging to Tric h oderma genus, by culturing, it is possible to manufacture a highly cellulase active enzyme of the present invention.

  Moreover, the cellulase activity in this invention can be represented by the enzyme (CMC-ase) activity which decomposes | disassembles water-soluble cellulose (carboxymethylcellulose sodium salt), and can be measured by the method described below. The higher the CMC-ase activity, the better the cellulose degradation rate and rate. Moreover, the enzyme activity which decomposes | disassembles a cellooligosaccharide into glucose can be represented as (beta) -glucosidase ((beta) -Glucosidase) activity.

The present invention is able to increase the selectivity of cellooligosaccharides by using cellulase having high CMC-ase activity and low β-Glucosidase activity and highly controlled cellulase for enzymatic degradation of cellulosic substances. It has an outstanding effect.
Each enzyme activity is demonstrated below.

  The β-Glucosidase activity in the present invention is an enzyme activity that degrades cellobiose and generates glucose, and is quantified by β-Glucosidase activity (U / mL). In order to obtain cellooligosaccharides in a high yield, it is efficient to use the above-mentioned enzyme solution having a high CMC-ase activity and a low β-Glucosidase activity. The lower the β-Glucosidase activity, the more the degradation of cellooligosaccharides such as cellobiose is suppressed. A preferable range is 8.0 U / mL or less. More preferably, it is 5.0 U / mL or less, More preferably, it is 4.0 U / mL or less, Most preferably, it is 2.0 or less.

  The CMC-ase activity of the present invention refers to an enzyme activity that degrades cellulose and is measured as an enzyme activity (U / mL) that degrades carboxymethylcellulose sodium salt as a substrate. Higher CMC-ase activity is preferable because decomposition of cellulose is promoted. For this reason, the cellulase of the present invention is required to have this activity. A preferable range is 4.0 U / mL or more. A more preferable range is 6.0 U / mL or more, a further preferable range is 8.5 U / mL or more, and a particularly preferable range is 9.5 U / mL or more.

The method for measuring CMC-ase activity and β-Glucosidase activity of the present invention is shown below.
(1) CMC-ase activity 480 μl of 1% carboxymethylcellulose sodium salt solution dissolved in 20 mM acetic acid-sodium acetate buffer (pH 5) is prepared. To this, 20 μl of an appropriately diluted enzyme solution is added and reacted at 40 ° C. for 30 minutes. After stopping the reaction by heating at 95 ° C. for 15 minutes, the reducing sugar is colorimetrically determined by the 3,5-dinitrosalicylic acid method. A standard curve is prepared using a cellobiose standard solution, and the amount of enzyme that releases 1 μmol of reducing sugar per minute in terms of cellobiose is defined as one enzyme unit (1 U).

(2) β-Glucosidase activity 0.3 ml of enzyme solution was added to 0.3 ml of 2.0 mass% cellobiose (manufactured by Aldrich: special grade) dissolved in 200 mM acetic acid-sodium acetate buffer (pH 5), After reacting at 40 ° C. for 30 minutes and heating at 95 ° C. for 15 minutes to stop the reaction, a glucose quantification kit (glucose CII-Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.), which is an enzymatic method combining mutarotase and glucose oxidase ) To determine the glucose concentration in the reaction solution. The amount of enzyme that liberates 1 μmol of glucose per minute is defined as 1 enzyme unit (1 U).

Below, the selective suppression method of (beta) -Glucosidase is demonstrated.
In the method for producing cellulase with suppressed β-Glucosidase activity according to the present invention, it is necessary to suspend or dissolve cellulase in an aqueous medium. The cellulase used here may contain a medium component and an excipient in addition to the enzyme component involved in the enzymatic degradation of cellulose. In addition to a water-soluble component such as an enzyme, a water-insoluble component may be included. The concentration at which cellulase is suspended or dissolved in an aqueous medium is not particularly limited, but in order to enhance the effect of subsequent pH adjustment and heat treatment, at least the enzyme component is preferably dissolved. A preferable concentration range is 0.001% by mass or more. Moreover, an upper limit is 99 mass% or less. A more preferable range is 0.1% by mass to 30% by mass, and a particularly preferable range is 0.5% by mass to 10% by mass.

  In the present invention, the pH of the cellulase suspension or aqueous solution needs to be 7 or more. By setting the pH to 7 or more, β-Glucosidase activity can be suppressed. The higher the pH, the lower the β-Glucosidase activity. However, when the pH is too high, the cellulase activity represented by CMC-ase is reduced. A preferable pH range is 10 or less, more preferably 9 or less, and particularly preferably 8 or less.

  Here, in order to control the pH of the suspension or the aqueous solution, a pH adjusting agent such as an acid, an alkali, or a salt can be used. Although there is no restriction | limiting in particular about these types, As acids, carboxylic acids, such as protonic acids, such as hydrochloric acid, a sulfuric acid, and phosphoric acid, formic acid, and an acetic acid, can be used conveniently. Moreover, as an alkali, sodium hydroxide, sodium acetate, potassium hydroxide, sodium dihydrogen phosphate, etc. can be used conveniently. Further, salts may be used in combination. Acids, alkalis and salts used here are classified as pH adjusters in, for example, “Pharmaceutical Additives Encyclopedia” (published by Yakuji Nippo Co., Ltd.) and “Food Additives Official Document” (published by Yodogawa Shoten) Can be mentioned. Even if it uses individually by 1 type chosen from the above, it is also free to use 2 or more types together.

  The concentration of these pH adjusting agents is not particularly limited, but is preferably adjusted to an appropriate range in order to obtain the effects of the present invention. Preferably it is 1 mM-10M, More preferably, it is 10 mM-1M, Most preferably, it is 500 mM or less.

  In the present invention, the heating temperature of the cellulase suspension or aqueous solution is 30 ° C. or higher. The higher the heating temperature, the higher the effect of suppressing β-Glucosidase. The upper limit is not particularly set, and is adjusted as appropriate so that the CMC-ase activity does not decrease in the above-mentioned pH range. There is no limit to the pressure. However, the time required for the heat treatment needs to be adjusted to such an extent that cellulase is not deactivated.

  In the production method of the present invention, it is preferable that a cellulosic substance coexists with a cellulosic material. By allowing the cellulose-based substance to coexist, cellulase deactivation can be suppressed. The density | concentration of a cellulosic substance should just be 0.01 mass% or more, and the upper limit is not set, but it is 99 mass% or less practically.

  The cellulosic material used here may be water-soluble or water-insoluble. Examples of the cellulose material include wood, bamboo, wheat straw, rice straw, corn cob, cotton, ramie, bagasse, kenaf, beet, squirt, and bacterial cellulose. In addition to the natural cellulosic material produced by the above-mentioned animals and plants, the present invention includes a regenerated cellulosic material obtained by chemically and physically dissolving or swelling the natural cellulosic material once and then regenerating it. Further, a cellulose derivative material obtained by chemically modifying a cellulose material may be used. Industrially, these cellulose materials are natural cellulose materials such as pulp, cellulose powder and crystalline cellulose, regenerated cellulose materials such as rayon, various regenerated cellulose materials such as alkali cellulose and phosphoric acid swollen cellulose, carboxymethylcellulose sodium. Any of various cellulose derivatives such as salt, cellulose acetate, cellulose nitrate, cellulose acetate / succinate, methylcellulose, and ethylcellulose may be used. However, in order to use the obtained cellooligosaccharide for pharmaceuticals, foods, and cosmetics, it is more preferable to use natural cellulosic materials. Even if one kind of cellulosic material is used as a raw material, a mixture of two or more kinds can be used.

  As described above, when the cellulosic substance is allowed to coexist, it is preferable that the pH of the cellulase suspension or aqueous solution is set to 7 or less and stored for a certain period of time before heating. In addition, through this operation, the deactivation of cellulase can be suppressed.

In the production method of the present invention, the heating operation may be under stirring or standing. The stirring state is appropriately adjusted to such an extent that β-Glucosidase is suppressed and cellulase activity does not decrease.
This heat treatment may be performed batchwise or continuously.

  The aqueous solution mainly composed of cellulase obtained by the above heat treatment can be subjected to a purification treatment such as decolorization, desalting, enzyme removal, etc., if necessary. The purification method is not particularly limited as long as it is a known method. For example, activated carbon treatment, ion exchange resin treatment, chromatography treatment, microfiltration, ultrafiltration, reverse osmosis filtration and other filtration treatments, crystallization treatment, etc. are used. These may be used alone or in combination of two or more.

  The aqueous solution mainly composed of cellulase purified by the above method can be used as it is, but if necessary, it may be solidified by drying. The drying method is not particularly limited as long as it is a known method, but for example, spray drying, freeze drying, drum drying, thin film drying, shelf drying, airflow drying, vacuum drying, etc. may be used, and these may be used alone. You may use, or may combine 2 or more types.

A method for producing cellooligosaccharide will be described below.
Enzymatic degradation of cellooligosaccharides by cellulase may be a known method and is not particularly limited. For example, a cellulosic substance is suspended in an aqueous medium as a substrate, and the method of the present invention is used. Examples include a method in which the produced cellulase is added and heated to perform a saccharification reaction while stirring or shaking.

  In the above method, the suspension method, the stirring method, the addition method / order of addition of cellulase / substrate, the concentration thereof, and other reaction conditions are appropriately adjusted so that the cellooligosaccharide can be obtained in a higher yield. In this case, the pH and temperature of the reaction solution may be within the range where the enzyme is not deactivated. Generally, when the reaction is performed at normal pressure, the temperature is 5 to 95 ° C., and the pH is 1 to 11. Range may be sufficient.

  The cellulosic material used in the present invention may be water-soluble or water-insoluble, and its origin may be plant or animal. Examples of animals and plants that produce it include wood, bamboo, wheat straw, rice straw, corn cob, cotton, ramie, bagasse, kenaf, beet, squirts, and bacterial cellulose. In addition to the natural cellulosic material produced by the above-mentioned animals and plants, the present invention includes a regenerated cellulosic material obtained by chemically and physically dissolving or swelling the natural cellulosic material once and then regenerating it. Further, a cellulose derivative material obtained by chemically modifying a cellulose material may be used. Industrially, these cellulose materials are natural cellulose materials such as pulp, cellulose powder and crystalline cellulose, regenerated cellulose materials such as rayon, various regenerated cellulose materials such as alkali cellulose and phosphoric acid swollen cellulose, carboxymethylcellulose sodium. Any of various cellulose derivatives such as salts may be used. However, in order to use the obtained cellooligosaccharide for pharmaceuticals, foods, and cosmetics, it is more preferable to use natural cellulosic materials. Even if one kind of cellulosic material is used as a raw material, a mixture of two or more kinds can be used.

  This enzyme reaction may be performed in a batch system or a continuous system. In order to avoid product inhibition by cellobiose in the enzymatic degradation reaction, it is necessary to maintain the cellobiose concentration in the reaction system within a specific range so that cellulase product inhibition is not affected. It is important in improving. As a method of keeping the cellobiose concentration in the reaction system within a specific range, a method of extracting cellobiose generated from the reaction system by membrane filtration such as ultrafiltration or reverse osmosis filtration may be used. Dry plant powder such as activated carbon, bamboo, and wood Alternatively, an organic porous substrate such as silicon dioxide or an inorganic porous substrate such as silicon dioxide may be introduced into the reaction system, and cellobiose may be adsorbed to the reaction system. A cellulose substrate is fixed to a column or the like, and a reaction solution containing cellulase is prepared. The method of making it distribute | circulate may be sufficient and the method of fix | immobilizing a cellulase to a polymer etc. and distribute | circulating the reaction liquid containing a cellulose may be sufficient.

  The aqueous solution mainly composed of cellooligosaccharide obtained by the above-described enzymatic decomposition can be subjected to purification treatment such as decolorization, desalting, and enzyme removal, as necessary. The purification method is not particularly limited as long as it is a known method. For example, activated carbon treatment, ion exchange resin treatment, chromatography treatment, microfiltration, ultrafiltration, reverse osmosis filtration and other filtration treatments, crystallization treatment, etc. are used. These may be used alone or in combination of two or more.

  The aqueous solution mainly composed of cellooligosaccharide purified by the above method can be used as it is, but if necessary, it may be solidified by drying. The drying method is not particularly limited as long as it is a known method, but for example, spray drying, freeze drying, drum drying, thin film drying, shelf drying, airflow drying, vacuum drying, etc. may be used, and these may be used alone. You may use, or may combine 2 or more types.

  As the cellooligosaccharide medium during the above purification and drying treatment, an organic solvent or the like may be used as necessary in addition to water. Although it does not restrict | limit especially also in the organic solvent used here, For example, what is used in the process of manufacturing a pharmaceutical, foodstuffs, and those additives is preferable, and "pharmaceutical additive encyclopedia" (Pharmaceutical Daily Inc.) Issued), “Japanese Pharmacopoeia” and “Food Additives Official Document” (both published by Yodogawa Shoten). Water and organic solvents can be used alone or in combination of two or more, and once dispersed in one medium, the medium is removed and dispersed in a different medium. Also good.

  Although there is no restriction | limiting in particular in the form of the cellooligosaccharide which passed through said process, For example, it can be used with solid, a suspension, an emulsion, a syrup, and a solution form at normal temperature. Examples of solid cellooligosaccharides include powders, granules, pellets, molded bodies, laminates, and solid dispersions.

  The use of cellooligosaccharides produced by the method of the present invention is not particularly limited. For example, in the fields of food, cosmetics, pharmaceuticals, general industrial products, etc., food ingredients, cosmetic ingredients, pigment ingredients, perfume ingredients, pharmaceutical medicinal ingredients , Agricultural chemical ingredients, feed ingredients, fertilizer ingredients, medium ingredients, analytical reagent ingredients, and additives, intermediate raw materials, fermentation raw materials and the like.

  Cellooligosaccharides produced by the method of the present invention are foods such as gels such as jelly, pudding and yogurt, seasonings such as mayonnaise, dressings, sauces, sauces, soups, processed vegetable products, curry, hayashi, Retort food such as meat sauce, stew, soup, chilled food, hamburger, bacon, sausage, salami sausage, hams and other livestock products, salmon, chikuwa, fish ham / sausage, fried rice cakes, bread, raw noodles , Dried noodles, macaroni, spaghetti, Chinese bun peel, cake mix, premix, white sauce, dumplings, spring rolls and other processed wheat foods, curry, sauce, soup, boiled canned jams, jars, etc. Candy, troche, tablet confectionery, chocolate, biscuits, cookies, rice confectionery, Japanese and Western confectionery, Western confectionery, Used in pastries such as nack confectionery, sugar confectionery, pudding, fried foods, croquettes, dumplings, Chinese buns and other processed foods, vegetable paste, meat mince, fruit paste, seafood paste, etc. . Moreover, it is used for dairy products such as ice cream, ice milk, lact ice, whipped cream, condensed milk, butter, yogurt, cheese, white sauce, and processed oils and fats such as margarine, fat spread, and shortening. In addition, carbonated drinks such as cola, carbonated drinks, alcohol drinks, fruit drinks mixed with dairy products, fruit juice or drinks containing fruits, milk drinks, coffee, milk, soy milk, cocoa milk, fruit milk, yogurt, etc. May be used for tea beverages such as lactic acid / milky beverages, sencha, oolong tea, matcha tea, black tea, and the like.

  Cellooligosaccharides produced by the method of the present invention are useful for intestinal useful bacterial flora such as activation of lactic acid bacteria, lactobacilli, etc., blood sugar concentration, blood insulin concentration reduction, blood cholesterol reduction, body fat In addition to the above normal food applications, functional foods and health foods can be used in addition to the above normal food applications. It may also be used in applications such as diet foods.

In addition, since the cellooligosaccharide produced by the method of the present invention has high purity, it may be used as a raw material for chemical conversion to various cellooligosaccharide derivatives.
The present invention will be described more specifically based on examples, but the present invention is not limited to these examples.

The Tric h oderma reesei NBRC31329 strain on a potato dextrose agar slant medium, 28 ° C., and cultured for 7 days to sufficiently form spores. Part 1 of the platinum ear was 10.0 g of cellobiose, 1.0 g of polypeptone, 0.5 g of yeast extract, 2.0 g of KH ₂ PO ₄ , 1.5 g of (NH ₄ ) ₂ SO ₄ , 0.3 g of MgSO ₄ .7H ₂ O, CaCl ₂ · _{2H 2 O0.3g,} Tween 80 [Nakalai chemicals Co., Ltd.] 1.0 ml, trace elements solution _{(H 3 BO 4 6mg, (} NH 4) 6 Mo 7 O 24 · 4H 2 O26mg, FeCl 3 · Dissolve and suspend 1.0 ml of 6H ₂ O 100 mg, CuSO ₄ · 5H ₂ O 40 mg, MnSO ₄ · 4H ₂ O 8 mg, ZnSO ₄ · 7H ₂ O 200 mg dissolved in 100 ml of water and suspended in 1 ml of water. , Adjusted to pH 5.0, dispensed 100 mL into a 500 mL Erlenmeyer flask and inoculated into autoclaved medium, 28 ° C., 6 days Cultured with shaking.

Further, in the above culture method, cellobiose was changed to glucose, and the cells were similarly cultured for 6 days.
These culture solutions were mixed, centrifuged, and 10 ml was concentrated to 1.5 ml by ultrafiltration to obtain a crude enzyme solution. (The CMC-ase of this crude enzyme solution was 12.18 U / mL, and β-Glucosidase was 23.9 U / mL.)

[Examples 1 to 5]
The crude enzyme solution obtained in Production Example 1 was dissolved in a 100 mM phosphate buffer having a pH of 7.0, pH 8.1, or pH 9.2 and stored at 30 ° C. or 60 ° C. for 30 minutes, and then CMC-ase. Activity, β-Glucosidase activity was measured. Table 1 shows the results obtained. The residual rate of activity was determined by the following formula. Activity remaining rate (%) = activity after storage (U / mL) / activity before storage (U / mL) × 100.

  From Table 1, each Example adjusted to pH 7 or more maintained 40% or more activity as CMC-ase, and remained 30% or less activity as β-Glucosidase.

[Comparative Example 1]
After the crude enzyme solution obtained in Production Example 1 was stored at pH 8.1 and 10 ° C. for 30 minutes, the residual activity rates of CMC-ase and β-Glucosidase were measured in the same manner as in the Examples. The results are shown in Table 1. In Comparative Example 1, although CMC-ase was maintained, β-Glucosidase was not significantly reduced.

[Comparative Example 2]
After the crude enzyme solution obtained in Production Example 1 was stored at pH 6.5 and 10 ° C. for 30 minutes, the residual activity rates of CMC-ase and β-Glucosidase were measured in the same manner as in the Examples. The results are shown in Table 1.
In Comparative Example 2, as in Comparative Example 1, CMC-ase was maintained, but β-Glucosidase was not significantly reduced.

[Comparative Example 3]
After the crude enzyme solution obtained in Production Example 1 was stored at pH 6.0 and 50 ° C. for 30 minutes, the residual activity rates of CMC-ase and β-Glucosidase were measured in the same manner as in the Examples. The results are shown in Table 1.
In Comparative Example 3, as in Comparative Examples 1 and 2, CMC-ase was maintained, but β-Glucosidase was not significantly reduced.

Example 6
A commercially available cellulase (joint sake refined GODO-TCD) was dissolved in purified water at a concentration of 1% by mass, and after heat treatment in the same manner as in Example 2, the residual rate of CMC-ase activity and the residual β-Glucosidase activity The rate was measured. The residual rate of CMC-ase was 61%, and the residual rate of β-Glucosidase was 12%.

Example 7
400 μL of the enzyme solution after heat treatment obtained in Example 2 was subjected to enzymatic decomposition at 40 ° C. for 18 hours using a dispersion of 5% crystalline cellulose (Asahi Kasei Chemicals, Theolas PH-101) in 100 mM acetate buffer. went. After enzymatic degradation, the sugar composition was measured by HPLC. As a result, the cellobiose concentration was 0.10% by mass, the glucose concentration was 0.04% by mass, and the selectivity was 71% (cellobiose concentration (mass%) / (cellobiose concentration (mass%) + glucose concentration (mass). %))) And a high value.

[Comparative Example 4]
In the same operation as Example 7, the enzyme reaction was performed using the crude enzyme solution of Comparative Example 2. As a result, the cellobiose concentration was 0.19% by mass, the glucose concentration was 0.21% by mass, and the selectivity was as low as 47.5%.

Example 8
In the same heat treatment as in Example 2, 1% crystalline cellulose was allowed to coexist and heat treatment was performed in the same manner as in Example 2. After the heat treatment, the solid content was centrifuged, and CMC-ase activity and β-Glucosidase activity were measured for the supernatant in the same manner as in Example 2. The residual rate of CMC-ase activity was 52%, and the residual rate of β-Glucosidase activity was 1.5%. Inactivation of CMC-ase was suppressed by allowing the cellulose-based material to coexist with the heat treatment.

  The present invention relates to a method for producing cellulase in which β-Glucosidase activity is selectively suppressed to efficiently obtain cellooligosaccharides. The cellulase obtained by this method can be suitably used for the production of cellooligosaccharides.

Claims (2)

(I) the cellulase obtained from Trichoderma reesei (Trichoderma reesei) suspended or dissolved in an aqueous medium, comprises heating the pH of the suspension or solution 7 above to the 30 ° C. or higher, beta- A method for producing cellooligosaccharide, comprising a step of producing a cellulase in which glucosidase activity is suppressed , and a step of decomposing a cellulosic substance using the cellulase obtained in the step (i). The method for producing cellooligosaccharide according to claim 1, wherein in the step (i), the suspension or the aqueous solution is heated to 30 ° C or higher in the presence of 0.01% by mass or more of a cellulose-based substance.