JP4789501B2 - Method for producing cellulase - Google Patents

Description

  The present invention relates to a method for producing cellulase, which easily obtains a cellulase with low β-glucosidase activity contamination without going through a special purification step. Furthermore, the present invention relates to a method for producing cellooligosaccharide, which uses cellulase with low β-glucosidase activity and selectively produces cellooligosaccharide in high yield.

Cellooligosaccharide is a general term for cellobiose, cellotriose, cellotetraose, cellopentaose and cellohexaose, and is a variety of sugars in which 1 to 6 glucopyranose units are linked by β-1,4.
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 producing cellooligosaccharide, in the enzymatic degradation system of cellulose, cellooligosaccharide obtained as a degradation product is further decomposed into glucose units by β-glucosidase which is an enzyme component in cellulase. The problem is that the yield decreases (see Non-Patent Document 2).
In the past, many attempts have been made to improve the yield of cellooligosaccharides during the enzymatic degradation of cellulose.

Examples of methods for selectively obtaining cellooligosaccharides by using cellulase and enzymatically decomposing cellulose include the following.
In Patent Document 1, a cellulose raw material containing a large amount of non-crystalline cellulose is used, and a hydrolysis reaction with cellulase is performed in the presence of lignin, and at least cellobiose among cellooligosaccharides produced by the hydrolysis reaction is occasionally reacted. A method for producing cellooligosaccharides collected from potato is disclosed. In Patent Document 2, wet pulp obtained by digesting a raw material containing natural lignocellulose and not drying after digestion is partially hydrolyzed with cellulase to obtain cellooligosaccharides. Among them, a method for producing a cellooligosaccharide that collects at least cellobiose is disclosed.

These production methods adsorb β-glucosidase, which is a cellooligosaccharide degrading enzyme contained in cellulase, to lignin and inhibit the action of β-glucosidase, thereby suppressing the decomposition of cellooligosaccharide into glucose. The reaction selectivity can be increased. However, in these production methods, since a large amount of lignin is contained in the obtained saccharified solution, the yield of cellooligosaccharide is reduced as a result. In addition, in order to obtain a high-purity cellooligosaccharide, delignification treatment from a saccharified solution is required, so that there is a problem that the purification process becomes complicated.
Patent Document 3 discloses a method for producing cellobiose, which is a kind of cellooligosaccharide, by reacting lignocellulose containing 1-20% by mass of lignin together with cellulase and lignin-degrading bacteria such as white rot fungi. Yes. In this production method, the action of cellulase on the substrate can be enhanced without undergoing delignification treatment of cellulose. However, since the degradation product contains lignin degradation products in addition to cellobiose, cellooligosaccharides are used in the same manner as described above. Yield is low. In addition, in order to obtain high-purity cellobiose, there is a problem that a lignin degradation product removal step is required, and the purification step becomes complicated.

Moreover, the following are mentioned as a method of improving the yield of cellooligosaccharide by using specific cellulase and carrying out the enzymatic decomposition of cellulose.
In Patent Document 4, product inhibition is achieved by combining an ultrafiltration reactor in a method for producing cellooligosaccharides from a cellulosic substance in an aqueous reaction solution by the action of cellulase produced by a microorganism belonging to the genus Cellobibrio. A method for producing a cellooligosaccharide that is released to accumulate and accumulate cellooligosaccharide is disclosed. According to this method, cellooligosaccharides composed only of cellobiose and cellotriose can be obtained as a decomposition product of enzymatic decomposition of the cellulosic material. However, enzymes produced by microorganisms belonging to the genus Cellobiblio are less likely to act on crystalline cellulose, and in order to shorten the reaction time and improve yield, amorphous cellulose is required as a substrate, and the process is complicated. There was a problem of becoming.

  In Patent Document 5, in the 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 such a production method, glucose can be reduced by enzymatic decomposition of cellulose, and a degradation product having 60% or more of 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.

  In Patent Document 6, cellulase is dissolved in cellulase by dissolving cellulose ester or cellulose ether ester or both and cellulase, keeping the temperature constant for a certain period of time, and then changing the pH to separate the insolubilized solid fraction from the solution. A cellulase purification method for selectively removing β-glucosidase contained in the solution, and cellulase from which β-glucosidase has been removed and cellulose are added to an aqueous medium to form a suspension. A method for producing cellobiose is disclosed in which cellobiose is collected by keeping the temperature for a period of time to produce cellobiose in the suspension.

Patent Document 7 discloses a solid fraction obtained by dissolving the chitosan and cellulase in an aqueous medium obtained by adjusting the pH so that chitosan is soluble, keeping the temperature constant for a certain period of time, and then changing the pH to insolubilize. A cellulase purification method that selectively removes β-glucosidase contained in cellulase by separating the solution, and cellulase and cellulose from which β-glucosidase has been removed are added to an aqueous medium and suspended. A method for producing cellobiose is disclosed in which the suspension is kept warm for a certain period of time, cellobiose is produced in the suspension, and the cellobiose is collected.
According to these production methods, the yield of cellobiose is improved by subjecting cellulase to adsorption separation treatment with a cellulose derivative or chitosan, and contacting with cellulose in a state of being adsorbed on the cellulose derivative or chitosan. However, since this production method requires purification of cellulase, the production process is complicated, and the cellulose derivative and chitosan used for cellulase purification are expensive, and there is a problem of high cost. Moreover, since cellulase is used for cellulose enzyme decomposition together with cellulose derivatives or chitosan, there is a problem that a step of removing them from the decomposition reaction solution is required.

Cellulose Communications, 5, No 2, 91-97 (1998) "Cellulase" Kodansha Scientific issue, 97-104 (1987) JP 05-317073 A Japanese Patent Application Laid-Open No. 07-184678 Japanese Patent Laid-Open No. 08-089274 Japanese Patent Laid-Open No. 01-256394 JP 05-115293 A JP 05-227957 A Japanese Patent Laid-Open No. 05-227958

  The present invention uses cellulosic material as a raw material and is enzymatically decomposed in the presence of cellulase, so that cellulase useful for producing cellooligosaccharide selectively and in high yield can be easily obtained without going through the enzyme purification step. The present invention also provides a method for producing cellulase.

The present inventors have found a method for producing cellulase in which cellulase-producing bacteria are cultured under pH control to reduce by-products of glucosidase.
Furthermore, the present inventors have found a method for selecting a strain having low β-glucosidase activity from cellulase-producing bacteria and succeeded in obtaining a strain in which β-glucosidase by-product is reduced from the mutant by, for example, inducing mutation.
That is, the present invention is as follows.
(1) Cellulase is produced by culturing Trichoderma reesei NBRC31329 strain, which is a cellulase-producing bacterium, at a pH of less than 3.5, and the resulting cellulase is used to enzymatically decompose cellulose. A method for producing cellooligosaccharide, which is characterized.
(2) The cellulase-producing bacterium is a Trichoderma reesei NBRC31329 mutant strain, Trichoderma reesei GL-1 (National Institute of Advanced Industrial Science and Technology, Patent Biological Depositary Accession Number FERM BP- 10323) The method for producing a cellooligosaccharide according to (1) above, wherein

  According to the present invention, in the method for producing cellooligosaccharides by enzymatic degradation of cellulosic substances, cellooligosaccharides can be selectively produced in high yield.

The cellulase of the present invention is a general term for enzymes that decompose cellulose, and is included in the cellulase referred to in the present invention as long as it has cellulolytic activity. Examples of cellulase-producing bacteria include microorganisms belonging to the genus Trichoderma, the genus Acremonium, the genus Aspergillus, the genus Bacillus, etc., which are microorganisms that produce known cellulases. it can.
The cellulase obtained in the present invention has an activity ratio of β-glucosidase activity to cellulose degrading activity (β-glucosidase activity / crystalline cellulose degrading activity) of 0.35 or less, more preferably 0.30 or less, Preferably it is 0.25 or less. The activity ratio here is represented by the ratio of the cellulase crystalline cellulose resolution and the cellobiose resolution (β-glucosidase). The smaller the activity ratio is, the lower the oligosaccharide resolution and the better the productivity and selectivity of the oligosaccharide. The lower limit is not particularly limited, but the range of the activity ratio that can be easily achieved is 0.01. That's it.

The enzyme activity can be measured by the following method.
(1) Crystalline cellulose decomposition activity 5% by mass of crystalline cellulose suspended in 50 mM acetic acid-sodium acetate buffer (pH 5) (manufactured by Asahi Kasei Chemicals Co., Ltd., trade name: Seolus PH-101 as 60% moisture, manufactured by Sanei Seisakusho) , Trade name: 0.1 ml of an enzyme solution appropriately diluted to 0.4 ml of a substrate solution of a universal stirring mixer kneaded and stirred at 126 rpm for 90 minutes with a hook blade, and then placed in a 40 ° C. water bath for 4 hours. After the reaction, the reaction is stopped by heating at 95 ° C. for 10 minutes, and the glucose concentration in the reaction solution is quantified by HPLC. The amount of enzyme in which the total amount of glucose and cellooligosaccharide released per minute is 1 μmole is defined as one enzyme unit (1 U).

(2) β-glucosidase activity 0.1 ml of enzyme solution was added to 0.4 ml of a substrate solution of 2.5% by mass of cellobiose (manufactured by Aldrich: special grade) dissolved in 50 mM acetic acid-sodium acetate buffer (pH 5), After reacting in a 40 ° C. water bath for 4 hours, the reaction is stopped by heating at 100 ° C. for 10 minutes, and the glucose concentration in the reaction solution is quantified by HPLC. The amount of enzyme that liberates 1 μmole of glucose per minute is defined as one enzyme unit (1 U).
In the various activity measuring methods described above, cellooligosaccharide and glucose in the reaction solution are quantified by high performance liquid chromatography (column: manufactured by Shimadzu Corporation, trade name: Asahipak NH ₂ P-50, high performance liquid chromatography: manufactured by Shimadzu Corporation, Product name: SCL-10A type, moving bed: acetonitrile / water = 75/25 (volume ratio) circulation rate: 1 ml / min. Sample solution: 10 μl).

A strain having a low (β-glucosidase activity / crystalline cellulose degradation activity) ratio can be obtained, for example, by the following method.
If necessary, microorganisms capable of producing cellulase are subjected to known mutagenesis treatments such as ultraviolet irradiation and the use of mutagens such as nitrosoguanidine, and from those strains (β-glucosidase activity / crystalline cellulolytic activity) Select strains with a low ratio. As a microorganism used for the mutagenesis treatment, for example, Trichoderma reesei NBRC31329 is used as a parent strain and cultured on a potato dextrose agar slope medium at 28 ° C. for 3 to 10 days. The produced spores are suspended in physiological saline to a concentration of 10 ⁵ to 10 ⁸ cells / ml, and subjected to mutation treatment with EMS (ethyl methyl slfonate) (100 to 500 μg / ml, pH 7.0, 28 ° C., 5 to 5 ° C.). 24 hours).
Selection of a strain with a low ratio of (β-glucosidase activity / crystalline cellulose degradation activity) is performed by collecting spores from this mutagenized spore suspension by centrifugation, washing well, culturing glucose as a carbon source, and culturing. This is achieved by measuring the enzyme activity of the product by a known method.
For example, by using a culture of each mutant-treated strain, enzymatic degradation using cellobiose or crystalline cellulose as a substrate, and the resulting reducing sugar may be quantified. By using a known chromogenic substrate, an enzymatic reaction with the culture is performed. A target strain may be selected qualitatively.

A method for obtaining cellulase from a cellulase-producing bacterium is obtained by culturing the strain and obtaining a culture supernatant. As the carbon source used in the medium, cellulose powder, cellobiose, filter paper, general paper, sawdust, bran, rice bran, bagasse, soybean meal, coffee cake, starch, lactose, and the like are used. As the nitrogen source, inorganic nitrogen salts such as ammonium sulfate and ammonium nitrate, organic nitrogen-containing substances such as urea, amino acids, meat extract, yeast extract, polypeptone, and protein degradation products are used. As inorganic salts, KH ₂ PO ₄ , MgSO ₄ .7H ₂ O, CaCl ₂ .2H ₂ O, FeCI ₃ .6H ₂ O, MnCl ₃ .4H ₂ O, ZnSO ₄ .7H ₂ O, etc. are used. If necessary, a medium containing organic micronutrients is used. A normal aeration and agitation culture apparatus is used for culturing, and the medium is used to control the temperature at which the production strain can grow and around the pH at which it can grow. Subsequently, the cells are removed from the culture solution by a known method such as centrifugation or filtration to obtain a supernatant. This supernatant can be used as a crude enzyme solution as it is.

Further, as a culture method for further reducing the (β-glucosidase activity / crystalline cellulose degradation activity) ratio, for example, during the culture, the pH of the culture solution is controlled to be less than pH 3.5 and higher than the pH at which cellulase-producing bacteria can grow. A method is mentioned. Specifically, the above-mentioned Trichoderma reesei NBRC31329 strain or its mutant strain is cultured at 25-35 ° C. for 3-10 days on a potato dextrose agar slope, cultured and suspended, dissolved, and a medium containing cellulose as a carbon source has a volume of 500 ml. Dispense 100 ml into an Erlenmeyer flask, inoculate into a medium sterilized by autoclave, and perform preculture at 28 ° C. for 2 to 4 days. Furthermore, 3 l of the same medium was placed in a 5 l jar fermenter and sterilized by autoclaving. The preculture was transplanted, cultured at 28 ° C. with stirring at 200 to 400 rpm and aeration 0.3 to 1 vvm, and NaOH was cultured. Alternatively, the pH is controlled to 2 to 3.5, preferably 2.5 to 3.0 with aqueous ammonia. After culturing for 4 to 7 days, the culture solution is removed by known methods such as centrifugation and filtration to obtain a supernatant. This supernatant can be used as a crude enzyme solution as it is.

A method for producing cellooligosaccharide will be described below.
The enzymatic decomposition method may be any known method, and is not particularly limited. For example, the cellulosic substance is suspended in an aqueous medium as a substrate, the cellulase of the present invention is added, and the mixture is stirred. Alternatively, a method of heating and carrying out a saccharification reaction may be mentioned.
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. Also, the pressure, temperature, for the pH, the same, but in which cellooligosaccharides is appropriately adjusted so that higher yields are obtained, the Trichoderma reesei NBRC31329 strain or a mutant strain of the above-described cellulase producing fungus, In the case of using the obtained cellulase, the enzymatic degradation of cellulose is preferably carried out at normal pressure and in an acetic acid or phosphate buffer solution at a temperature of 50 to 60 ° C. and a pH of 3.0 to 5.5.

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 important to maintain the cellobiose concentration in the reaction system within a specific range in order to improve the productivity of cellooligosaccharide. 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, wood, etc. 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 the cellooligosaccharide obtained by 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, culture medium ingredients, analytical reagent ingredients, and additives, intermediate raw materials, fermentation raw materials and the like.

  Cellooligosaccharides obtained by the present invention are used in foods, for example, gels such as jelly, pudding, and yogurt, mayonnaise, dressings, sauces, sauces, soups, processed vegetables and other seasonings, curry, hayashi, meat sauce, stew , Retort food such as soup, chilled food, hamburger, bacon, sausage, salami sausage, livestock products such as hams, water paste products such as salmon, chikuwa, fish ham and sausage, fried rice cake, bread, raw noodles, dry noodles, Processed wheat foods such as macaroni, spaghetti, Chinese bun skin, cake mix, premix, white sauce, gyoza and spring rolls, canned curry, sauce, soup, boiled, jam, bottling, candy, Lozenges, tablet confectionery, chocolate, biscuits, cookies, rice confectionery, Japanese and Western confectionery, Western confectionery, snacks Child, confectionery sugar confectionery, such as pudding, fries, croquettes, dumplings, cooking processed products such as Chinese bun, vegetable paste, meat of minced, fruit paste, is used, for example, paste such as seafood paste. 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 obtained in 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 percentage reduction In addition to the above normal food applications, functional foods, health foods, diet foods, etc. in addition to the above normal food applications It may be used for such purposes.
Moreover, since the cellooligosaccharide obtained by this invention is highly purified, you may use it as a chemical conversion raw material 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.
[Example 1]
Tricoderma reesei NBRC31329 is cultured on potato dextrose agar slant at 28 ° C. for 7 days. The produced spores are suspended in 2 ml of 100 mM calcium phosphate buffer (pH 7) so as to have a concentration of 10 ⁶ cells / ml, 24 ul of EMS (ethyl methanol slfonate) is added, and shaken at 28 ° C. for 16 hours to carry out a mutation treatment. Spores are collected from this spore suspension by centrifugation, washed well with 100 mM calcium phosphate buffer (pH 7), diluted to 100-300 spores per plate, 1 g glucose, 1 g yeast extract, (NH ₄ ) _2. SO ₄ 2 g, KH ₂ PO ₄ 4 g, Na ₂ HPO ₄ 2 g, MgSO ₄ .7H ₂ O 200 mg, CaCl ₂ .2H ₂ O 1 mg, Triton X-100, Trace element 1 ml (Boric acid 6 mg, Ammonium molybdate tetrahydrate 26 mg, iron chloride (3) hexahydrate 100 mg, copper sulfate pentahydrate 40 mg, manganese sulfate tetrahydrate 8 mg, zinc sulfate heptahydrate 200 mg dissolved in 100 ml of purified water in total) Dissolve or suspend 20 g of agar in 1 l of water, sterilize with an autoclave, sterilize by filtration with a membrane filter, and culture at 28 ° C. for 5 days. To measure the Koshidaze activity and crystalline cellulose degradation activity, was selected mutant strain GL-1 (National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, accession number FERM BP -10323) a.

[Example 2]
Each strain of Trichoderma reesei NBRC31329 and the mutant strain GL-1 obtained in Example 1 is cultured on a potato dextrose agar slope medium at 25 ° C. for 7 days to sufficiently form spores. Part 1 1.0 g of polypeptone, 0.5 g of yeast extract, KH ₂ PO ₄ 2.0 g, (NH ₄ ) ₂ SO ₄ 1.5 g, MgSO ₄ .7H ₂ O 0.3 g, CaCl ₂ .2H ₂ O 0.3 g , Tween 80 [manufactured by Hanai Chemicals Co., Ltd.] 1.0 ml, trace element liquid (H ₃ BO ₄ 6 mg, (NH ₄ ) ₆ Mo ₇ O ₂₄ · 4H ₂ O 26 mg, FeCl ₃ · 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 and suspended in 100 ml of water) 1.0 ml and tartaric acid 7.5 g dissolved and suspended in 1 l of water, pH 4. Adjust to 0, dispense 100 ml into a 500 ml Erlenmeyer flask, add 1 g of crystalline cellulose (trade name PH-101, manufactured by Asahi Kasei Chemicals), inoculate into a medium sterilized by autoclave, and shake at 28 ° C. for 5 days Cultured. On the fifth day, the culture solution was centrifuged, and the cellulase activity and β-glucosidase activity of the supernatant were measured. The results are shown in Table 1.

[Example 3]
Potato dextrose medium (Difco) is inoculated with Trichoderma reesei NBRC31329 and cultured at 37 ° C. for 7 days. One platinum spore was taken from the surface of the medium, 1 g polypeptone, 0.5 g yeast extract, 2 g potassium phosphate, 1.5 g ammonium sulfate, 0.3 g magnesium sulfate, 0.3 g calcium chloride, 1 ml trace element (6 mg boric acid, Ammonium molybdate tetrahydrate 26 mg, iron chloride (3) hexahydrate 100 mg, copper sulfate pentahydrate 40 mg, manganese sulfate tetrahydrate 8 mg, and zinc sulfate heptahydrate 200 mg were added to 100 ml of purified water. 1 ml of Adecanol LG-109 was suspended and dissolved in 1 l of purified water, and 100 ml was dispensed into a 500 ml Erlenmeyer flask. Crystalline cellulose (trade name: PH-101, manufactured by Asahi Kasei Chemicals) was placed in each flask. ) After adding 1g, inoculate the medium sterilized by autoclave and pre-culture at 28 ° C for 3 days Yeah. Furthermore, 30 ml of the preculture was transplanted to a 5 l jar fermenter charged with 3 l of the same medium, cultured at 28 ° C., stirring at 400 rpm and aeration of 0.5 vvm, and the lower limit of pH was controlled at pH 3.0 with NaOH aqueous solution during the cultivation. . After culturing for 5 days, the cultured solution was centrifuged to obtain a supernatant as a crude enzyme. Crystalline cellulase decomposing activity and β-glucosidase activity of the obtained enzyme solution were measured by the methods described above. Changes in pH over time during the culture are shown in FIG. 1, and the activity measurement results are shown in Table 2.

[Example 4]
When culturing Trichoderma reesei NBRC31329 in the same manner as in Example 3, the lower limit of pH was controlled at pH 2.5 with NaOH during the cultivation to obtain a crude enzyme solution. Crystalline cellulase decomposing activity and β-glucosidase activity of the obtained enzyme solution were measured by the methods described above. Changes in pH over time during the culture are shown in FIG. 1, and the activity measurement results are shown in Table 2.
[Comparative Example 1]
When culturing Trichoderma reesei NBRC31329 in the same manner as in Example 3, the lower limit of pH was controlled to pH3.5, pH4, or pH5 with NaOH during the cultivation to obtain a crude enzyme solution. Crystalline cellulase decomposing activity and β-glucosidase activity of the obtained enzyme solution were measured by the methods described above. Changes in pH over time during the culture are shown in FIG. 1, and the activity measurement results are shown in Table 2.

[Example 5]
The GL-1 strain obtained in Example 1 was cultured in the same manner as in Example 3. When culturing, a crude enzyme solution was obtained by controlling the lower limit of pH to pH 3 or pH 4 with NaOH during the culture. FIG. 2 shows changes with time in pH during the culture.
[Example 6]
5% by mass of crystalline cellulose (manufactured by Asahi Kasei Chemicals Co., Ltd., trade name: Theolas PH-101, 60% moisture, manufactured by Sanei Seisakusho, trade name: kneaded and stirred at 126 rpm for 90 minutes with a hook blade in a universal stirring mixer) 2 ml of the cellulase crude enzyme solution obtained in Example 3 and Example 5 was added to 8 ml, and hydrolysis was performed under a stirring condition at 55 ° C. After reaction for 2 hours, 4 hours, 6 hours, and 8 hours, heat at 95 ° C for 15 minutes to stop the enzyme reaction, obtain a supernatant by centrifugation, and measure the concentration of cellooligosaccharide and glucose by the HPLC method described above did. The results are shown in FIG.
[Comparative Example 2]
When enzymatically degrading crystalline cellulose in the same manner as in Example 6, the cellulase obtained in Comparative Example 1 was used as a crude enzyme solution. The results are shown in FIG.

  The cellooligosaccharides obtained by the production method of the present invention include, in addition to normal food materials, functional food materials, chemical conversion raw materials such as pharmaceuticals and other chemical intermediate synthesis materials, foods, pharmaceuticals, and general industrial products as fermentation raw materials. It can be suitably used in the field.

The time-dependent change of pH in the culture | cultivation of Example 3, 4 and the comparative example 1 is shown. The time-dependent change of pH in the culture | cultivation of Example 5 is shown. The relationship between the amount of cellulose degradation in the reaction liquid obtained by the enzymatic decomposition reaction of Example 6 and Comparative Example 2 and the purity of cellooligosaccharide is shown.

Claims (2)

A cellulase is produced by culturing a cellulase-producing bacterium Trichoderma reesei NBRC31329 strain at a pH of less than 3.5, and the resulting cellulase is used to enzymatically decompose cellulose. A method for producing cellooligosaccharides . The cellulase-producing bacterium is a Trichoderma reesei NBRC31329 mutant, Trichoderma reesei GL-1 (National Institute of Advanced Industrial Science and Technology, Patent Biodeposition Center Accession No. FERM BP- 10323) The method for producing a cellooligosaccharide according to claim 1.

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