JP6325214B2 - Cellulase production method and liquid medium - Google Patents

Description

  The present invention relates to a method for producing cellulase, and more particularly, to a method for producing cellulase by culturing Trichoderma using a liquid medium containing paper-derived pulp as a carbon source.

  In order to effectively use cellulose resources, in recent years, methods for efficiently decomposing cellulose have been searched. In nature, cellulose is mainly degraded by microorganisms, and it is known that various microorganisms such as bacteria and filamentous fungi produce cellulose-degrading enzymes.

  These microorganisms secrete cellulose-degrading enzymes outside the cells, and cellulose is decomposed into glucose mainly through cellooligosaccharides and cellobiose by its action. Cellulolytic enzymes are generally called cellulases.

  When artificially producing cellulase, Trichoderma is known as a microorganism that secretes cellulase and is widely used. A method of culturing a microorganism belonging to the genus Trichoderma (hereinafter referred to as “Trichoderma sp.”) Using a medium containing nutrients such as a carbon source and a nitrogen source to secrete cellulase is also known.

  Patent Document 1 describes a method for producing β-glucanase and xylanase by culturing Trichoderma spp. Using a liquid medium containing pulp derived from paper that is not subjected to heat treatment or alkali treatment as a carbon source. . Paper is easily available and inexpensive, and according to this method, highly active cellulase can be produced at low cost.

However, in order to implement on an industrial scale, it is still necessary to enhance the cellulase supply capacity, and a technique for stably increasing the production amount of cellulase is required.
International Publication No. 2010/70748

  In order to improve the production of cellulase, it is necessary to promote the growth of Trichoderma. For this purpose, it is effective to enrich the culture medium to enrich the nutrients of the genus Trichoderma. On the other hand, the medium needs to provide aeration during culturing and must have good agitation that can take up air.

  When paper contains water, it becomes pasty and increases in viscosity. Therefore, liquid media containing conventional pulp increase the content of papers and increase the concentration of the medium, thereby reducing the agitation and inhibiting the growth of Trichoderma sp. .

  Considering the stirring ability of a normally used aeration and stirring culture apparatus, the initial concentration of the liquid medium pulp is preferably about 5% or less. In this case, in order to increase the production amount of cellulase, it is necessary to add pulp to the liquid medium after the cultivation of Trichoderma has progressed to some extent. Adding pulp in the middle of the culture complicates the production process and also causes the liquid medium to be contaminated by various bacteria.

  The present invention solves the above-mentioned conventional problems. The object of the present invention is to provide a cellulase without complicating the production process in a cellulase production method for culturing Trichoderma using a liquid medium containing pulp. An object of the present invention is to provide a method for producing cellulase capable of increasing the production amount of

The present invention is a method for producing cellulase comprising a step of culturing Trichoderma using a liquid medium containing a water-soluble saccharide,
The liquid medium containing the water-soluble saccharide is obtained by adding cellulase to a paper-derived pulp that is not subjected to heat treatment or alkali treatment as a carbon source, and a raw liquid medium containing ammonia nitrogen or amino nitrogen as a nitrogen source, and an enzyme reaction And a method for producing cellulase, which is obtained by producing water-soluble saccharides.

  In one certain form, the density | concentration in the raw material liquid culture medium of the said pulp is 6% W / V or more.

  In one certain form, the density | concentration in the liquid culture medium containing the water-soluble saccharide of the said water-soluble saccharide is 1 weight% or more.

  In one certain form, the density | concentration in the raw material liquid culture medium of the said ammonia nitrogen or amino nitrogen is 35 mM or more.

  In one certain form, the said paper is at least 1 type selected from the group which consists of a quality paper, an additional paper, a copy paper, a newspaper, and a corrugated paper.

  In one certain form, the Trichoderma genus microbe is Trichoderma reesei.

  In one certain form, a pulp is not added with respect to the liquid medium containing a water-soluble saccharide in the process of culture | cultivation.

  In addition, the present invention is a water-soluble by adding cellulase to a pulp derived from paper not subjected to heat treatment or alkali treatment as a carbon source, and a raw material liquid medium containing ammonia nitrogen or amino nitrogen as a nitrogen source, followed by an enzymatic reaction. Provided is a liquid medium used for culturing Trichoderma spp. Obtained by producing saccharides.

  In one certain form, the density | concentration in the raw material liquid culture medium of the said pulp is 6% W / V or more.

  In one certain form, the density | concentration in the liquid culture medium containing the water-soluble saccharide of the said water-soluble saccharide is 1 weight% or more.

  In one certain form, the density | concentration in the raw material liquid culture medium of the said ammonia nitrogen or amino nitrogen is 35 mM or more.

  The present invention also provides a cellulase produced by any of the methods described above.

  Moreover, this invention provides the decomposition | disassembly or saccharification method of a cellulose resource characterized by using the said cellulase.

  According to the method of the present invention, in the cellulase production method for culturing Trichoderma using a liquid medium containing pulp, it is possible to increase the production amount of cellulase without complicating the production process. The cellulase obtained by the method of the present invention has high β-glucanase activity and xylanase activity, and is extremely useful for saccharification of natural cellulose resources containing xylan such as bagasse or rice straw.

Liquid Medium The liquid medium of the present invention is an aqueous liquid containing water-soluble saccharides and containing nutrients for growing Trichoderma spp. The liquid medium of the present invention is prepared using as raw materials a paper-derived pulp as a carbon source and a liquid medium (for example, Mandel medium) containing ammonia nitrogen or amino nitrogen as a nitrogen source. An example of a liquid medium (hereinafter referred to as “raw material liquid medium”) that is a raw material for preparing the liquid medium of the present invention is shown below.

Paper: 6 g, (NH ₄ ) ₂ SO ₄ : 0.14 g, KH ₂ PO ₄ : 1.5 g, CaCl ₂ · 2H ₂ O: 0.03 g, MgSO ₄ · 7H ₂ O: 0.03 g, corn steep liquor: 2 mL, Tween 80: 0.1 mL, trace elements solution _{(H 3 BO 4 6mg, (} NH 4) 6 Mo 7 O 24 · 4H 2 O 26mg, FeCl 3 · 6H 2 O 100mg, CuSO 4 · 5H 2 O 40 mg, MnCl ₂ .4H ₂ O 8 mg, ZnSO ₄ .7H ₂ O 200 mg solution): 0.1 mL, water: 100 mL (adjusted to pH 4.8 with phosphoric acid or sodium hydroxide)

  In order to prepare the liquid culture medium of the present invention, cellulase is added to the raw material liquid culture medium and subjected to an enzymatic reaction. The pulp hydrolyzes by reacting with cellulase. As a result, the viscosity of the liquid medium is reduced, and the stirring ability of the medium is improved. Further, the pulp is shortened and saccharified to produce short fibers, oligosaccharides, polysaccharides, low molecular sugars such as xylose, or other water-soluble saccharides.

  As a result of the enzyme reaction, the liquid medium of the present invention contains 0.1% by weight or more, preferably 0.5% by weight or more, more preferably 1% by weight or more of water-soluble saccharides. The water-soluble saccharide as used in the field of this invention means the water-soluble saccharide contained in the supernatant of a liquid medium. When the concentration of the water-soluble saccharide is 1% by weight or more, the production amount of cellulase is significantly improved.

  The upper limit of the concentration of the water-soluble saccharide is generally 10% by weight, preferably 7% by weight, more preferably 5% by weight. In order to increase the concentration of the water-soluble saccharide beyond 10% by weight, a large amount of enzyme or a long-time enzyme reaction is required, resulting in high cost or low production efficiency.

  The cellulase includes at least endoglucanase, exoglucanase, and β-glucosidase, and is not particularly limited as long as it has activity in a neutral to alkaline region, preferably pH 7 to 10. For example, a cellulase enzyme agent may be used. The amount of cellulase added is 1 to 200 V / W%, preferably 2.5 to 180 V / W%, particularly preferably 30 to 100 V / W%, based on the weight of the pulp.

  When the amount of cellulase added is less than 1 V / W% with respect to the weight of the pulp, a long time is required for the enzyme reaction, and the processing efficiency decreases. In addition, if the amount of cellulase added exceeds 200 V / W% with respect to the weight of the pulp, the treatment efficiency is not improved so much that a large amount of enzyme is required, so there is a problem in terms of cost.

  As for the reaction conditions for the enzyme reaction, the pH is preferably neutral to alkaline (pH 7 to 10) and the temperature is about 40 to 50 ° C. The reaction time is set so that the viscosity of the liquid medium containing pulp is lowered and the stirrability is improved. The reaction time is set so that the concentration of the water-soluble saccharide takes a preferable value in consideration of the amount of cellulase enzyme used. The reaction time of the enzyme reaction is, for example, 1 minute to 5 hours, preferably 5 minutes to 2 hours.

  Pulp refers to fibers used as raw materials for paper. The kind of the pulp is preferably one having high cellulose purity such as chemical pulp and waste paper pulp. A preferred pulp is a paper-derived pulp obtained by decomposing or cutting paper. That is, the pulp in the present specification refers to papers made intangible.

  Specific examples of preferable papers include high-quality paper, renewal paper, copy paper, newsprint, and cardboard paper. The papers only need to contain a preferable pulp, and may be printed or written or generally called waste paper. For example, old books, magazines and used notebook pages, flyers, envelopes, stationery, postcards, tissue paper, and the like can be used.

  The higher the concentration of the pulp in the raw material liquid medium, the better the enzyme production efficiency. That is, the upper limit is the limit amount at which the liquid medium after the enzyme reaction exhibits good agitation. In order to facilitate stirring and mixing of the raw material liquid medium, it is preferable to cut the paper with a shredder.

  The concentration of the pulp in the raw material liquid medium is 1% W / V or more, preferably 3% W / V or more, more preferably 6% W / V or more. When the pulp concentration is less than 1% W / V, the production amount of cellulase may not increase so much.

  When the enzymatic reaction with cellulase is not performed, a medium containing pulp at a concentration of 6% W / V or more has an increased viscosity and a lower stirrability. In that case, in a normally used aeration and agitation culture apparatus, air cannot be sufficiently entrained in the liquid medium, and if it is used as it is for culturing, there is a high possibility that the production amount of cellulase will decrease. When performing an enzyme reaction with cellulase, a medium containing pulp at a concentration of 6% W / V or more has a relatively short reaction time until the concentration of the water-soluble saccharide becomes 1% by weight or more.

  The concentration of the pulp in the raw material liquid medium can be 20% W / V or less, preferably 15% W / V or less, more preferably 10% W / V or less, depending on the performance of the stirring device. Generally, the concentration of pulp in the liquid medium as a raw material is 4 to 15% W / V, preferably 6 to 12% W / V.

  Pulp is a natural cellulosic material that retains the naturally occurring molecular structure. The raw material liquid medium of the present invention may use a natural cellulose material other than pulp as a carbon source in combination with or in place of pulp. Preferred natural cellulosic materials include beer lees, barley tea extract lees, wheat bran, apple pomace and the like. The production method and use method of these natural cellulose materials are specifically described in, for example, JP 2011-41540 A. The usage-amount of natural cellulose materials other than a pulp will not be specifically limited if it is the quantity of the grade which the liquid culture medium after an enzyme reaction shows favorable stirring property.

  Ammonia nitrogen means nitrogen contained in ammonia or ammonium salt derived from ammonia. The amino nitrogen means nitrogen contained in an amine or an amine-derived amino compound. The compound containing ammonia nitrogen or amino nitrogen is, for example, ammonium sulfate, ammonium nitrate, diammonium phosphate, ammonium chloride, aqueous ammonia, urea, amino acids and salts thereof (for example, sodium glutamate).

  Of these, a particularly preferred compound for use in the liquid medium of the present invention as a nitrogen source is ammonium sulfate. The reason is that the cost is low and it is easy to obtain.

  The concentration of ammonia nitrogen or amino nitrogen in the liquid medium is 35 to 660 mM as the number of moles of ammonium. Preferably, it is 50-580 mM. When the concentration is less than 35 mM, the production amount of cellulase, particularly β-glucanase may not increase so much. Further, when the initial concentration exceeds 660 mM, the productivity of the enzyme decreases. Moreover, it is preferable to increase / decrease the density | concentration in the liquid culture medium of ammonia nitrogen or amino nitrogen according to the density | concentration of the pulp in a liquid culture medium.

  The raw material liquid medium of the present invention may use waste microbial cells as a nitrogen source in combination with or in place of ammonia nitrogen or amino nitrogen. A waste cell refers to a residue such as a cell or a medium left after collection of a target component in production of a substance produced by growing a microorganism such as an enzyme, or a processed product thereof. A method for producing and using waste cells is specifically described in, for example, Japanese Patent Application Laid-Open No. 2011-41540. The amount of waste cells used is not particularly limited as long as the amount of cellulase produced is improved.

Method for producing cellulase Trichoderma is known as a cellulase producing bacterium required for saccharification of cellulose. The genus Trichoderma used in the present invention is not particularly limited as long as it produces cellulase. Preferred Trichoderma spp. Are Trichoderma reesei or Trichoderma viride. Particularly preferred is Trichoderma reesei.

  The mycological properties of the fungi Trichoderma reesei and Trichoderma viride are described, for example, by EG Simmons, Abst. 2nd International Mycological Congress, Tampa, Florida, USA (August 1977, p. 618).

  A normal aeration and agitation culture apparatus is used for liquid culture, and the culture temperature is 20 to 33 ° C., preferably 28 to 30 ° C., and the culture pH is 4 to 6 by using a liquid medium containing the water-soluble saccharide. Incubate for days.

  Then, if necessary, the cells are removed from the culture solution by a known method such as centrifugation or filtration to obtain a Trichoderma spp. Culture supernatant. The Trichoderma sp. Culture solution or culture supernatant contains a high concentration of the target cellulase, that is, β-glucanase and xylanase.

  The β-glucanase activity of the obtained culture solution or culture supernatant is 25 U / ml or more, preferably 40 U / ml or more, more preferably 50 U / ml or more. Moreover, the xylanase activity of this culture solution or culture supernatant is 60 U / ml or more, preferably 100 U / ml or more, more preferably 140 U / ml or more, and further preferably 160 U / ml or more. When either the β-glucanase activity or the xylanase activity of the culture solution or the culture supernatant falls below the lower limit, the effect on the purpose of effective use of various naturally occurring cellulose resources is reduced.

  The xylanase activity can be quantified by increasing the absorbance at 540 nm by reacting reducing sugar produced by enzymatic hydrolysis using oat spelts-derived xylan as a substrate.

  More specifically, 1% xylan substrate solution (Sigma, Xylan, from oat spelts dissolved in 200 nM acetate buffer (pH 4.5)) was added to 1.9 ml of the culture solution or culture supernatant solution 0.1 ml. After an enzyme reaction at 10 ° C. for exactly 10 minutes, DNS reagent (0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, 22.5% sodium potassium tartrate tetrahydrate, 0.3% lactose Add 4 ml (including monohydrate) and mix well to stop the reaction. In order to quantify the amount of reducing sugar contained in the reaction stop solution, the reaction stop solution is accurately heated in a boiling water bath for 15 minutes. Subsequently, after cooling to room temperature, the amount of reducing sugar corresponding to xylose is quantified by measuring the absorbance at 540 nm. One unit of xylanase activity is expressed as the amount of enzyme that produces reducing sugar corresponding to 1 μmol of xylose per minute under the reaction conditions of 40 ° C. and 10 minutes.

Cellulose Resource Decomposition or Saccharification Method The cellulase obtained by the method of the present invention is useful for decomposing or saccharifying cellulose resources. The cellulose resource here may be either synthetic cellulose or natural cellulose resources. Synthetic cellulose represents what is circulated as cellulose powder. Examples of natural cellulose resources include bagasse, rice straw, wheat straw, beer lees, and wood. Since the present invention can produce β-glucanase and xylanase at the same time, it is particularly excellent in saccharification of natural cellulose resources such as bagasse, rice straw, straw and beer lees.

  The method for decomposing or saccharifying the cellulose resource may be a known method, and is not particularly limited. For example, the cellulose resource is suspended in an aqueous medium as a substrate, and the above culture solution or culture medium is used. There is a method in which a saccharification reaction is performed by adding a clear liquid and heating the mixture while stirring or shaking. Instead of the above culture solution or culture supernatant showing cellulolytic activity, a dried product thereof or a solution obtained by dispersing or dissolving the dried product in water may be used.

  The cellulose raw material is preferably delignified in advance. The reaction conditions such as the suspension method, the stirring method, the method of adding the above mixed solution, the order of addition, and their concentrations are appropriately adjusted so that glucose can be obtained in a higher yield.

  In this case, the pH and temperature of the reaction solution may be within the range in which the enzyme is not inactivated. Range may be sufficient. Further, the pressure, temperature, and pH are adjusted as appropriate so that glucose can be obtained in a higher yield, as described above, but at normal pressure and in acetic acid or phosphate buffer, the temperature is 50-60. It is preferable to perform in the range of 4 degreeC and 6 degreeC. The reaction time is generally 6 to 147 hours, preferably 24 to 72 hours.

  By saccharification of cellulose, an aqueous solution containing glucose is obtained. The obtained aqueous solution can be subjected to purification treatment such as decolorization, desalting, enzyme removal, etc., 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 glucose purified by the above method can be used as it is, but may be solidified by drying as necessary. 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.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited to these Examples.

Reference example 1
(Pre-culture)
Trichoderma reesei QM9414 strain was cultured on potato dextrose (PD) agar medium at 28 ° C. for 7 days to sufficiently form spores. ^Six 1 × 10 ⁶ spores suspended in physiological saline were used as cellulose raw materials (just fiber: manufactured by International Fiber Corporation): 8 g, KH ₂ PO ₄ : 2 g, (NH ₄ ) ₂ SO ₄ : 1.4 g, polypeptone ( (DIFCO): 1 g, yeast extract (DIFCO): 0.5 g, CaCl ₂ · 2H ₂ O: 0.3 g, MgSO ₄ · 7H ₂ O: 0.3 g, Tween 80: 1 mL, trace element solution [H ₃ BO ₄ 6 mg, (NH ₄ ) 6Mo ₇ O ₂₄ · 4H ₂ O 26 mg, FeCl ₃ · 6H ₂ O 100 mg, CuSO ₄ · 5H ₂ O 40 mg, MnCl ₂ · 4H ₂ O 8 mg, ZnSO ₄ · 7H ₂ O 200 mg Solution dissolved and suspended in 100 ml of water]: Inoculate a 500 mL baffled Erlenmeyer flask containing 1 mL, shake culture at 28 ° C., 180 rpm, 3 days, To obtain a culture solution.

(Production of liquid medium containing water-soluble saccharides)
In a 500 mL Erlenmeyer flask with baffle, plain paper: 6 g, KH ₂ PO ₄ : 1.5 g, (NH ₄ ) ₂ SO ₄ : 1.68 g, CaCl ₂ · 2H ₂ O: 0.03 g, MgSO ₄ · 7H ₂ O : 0.03 g, Corn steep liquor: 2 mL, Tween 80: 0.1 mL, the above trace element solution: 0.1 mL, and water: 100 mL were prepared to prepare a raw material liquid medium. The plain paper was cut into 2 mm × 7 mm with a shredder (“Desk Parser DS-4100” manufactured by Carl) and used. A cellulase solution (“Cellulase SS” (trade name) manufactured by Nagase ChemteX, Inc., CMC activity: 1600 CNU / g) was added to this raw liquid medium at 2.5% V / V, and after shaking at 50 ° C. for 5 minutes, The enzyme reaction was stopped by heating in an autoclave. The water-soluble saccharide contained in the obtained liquid medium was decomposed into monosaccharides by the phenol-sulfuric acid method, and the concentration was measured. Details are shown below.

(Measurement of water-soluble sugar concentration)
The liquid medium collected as a sample was centrifuged (20000 G, 10 min: manufactured by Tommy Seiko Co., Ltd.), the supernatant was diluted to an appropriate magnification, and 500 mL of 5% phenol solution was added to 500 mL of the diluted solution, followed by stirring. Then 2.5 mL of concentrated sulfuric acid was added and stirred. After standing at room temperature for 20 minutes or more, the absorbance at 490 nm of the solution was measured with a spectrophotometer (model UV-1800, manufactured by Shimadzu Corporation). In addition, based on the standard produced with glucose, the weight% concentration of the water-soluble saccharide in each sample medium was calculated. Table 1 shows the measurement results of the water-soluble saccharide concentration.

(Main culture)
A 500 mL baffled Erlenmeyer flask containing a liquid medium containing a water-soluble saccharide was inoculated with 5 mL of the preculture solution, and cultured with shaking at 28 ° C., 180 rpm for 7 days. On the seventh day, the culture solution was centrifuged to obtain a supernatant. The enzyme activity of the obtained supernatant was measured by the operation described below. The measurement results of enzyme activity are shown in Table 1.

(Measurement of enzyme activity)
The β-glucanase activity was determined by measuring the absorbance of a stained fragment produced by enzymatic degradation using a β-glucan labeled with a substrate using a β-glucanase measurement kit manufactured by Megazyme. Specifically, 0.1 mL of the culture solution was added to 0.1 mL of the azo barley glucan substrate solution, and the enzyme reaction was performed at 40 ° C. for exactly 10 minutes, and then the stop solution [4% sodium acetate, 0.4% zinc acetate, 0.6% L containing 80% methyl cellosolve (pH 5)] was added and left for 5 minutes to stop the reaction. Subsequently, after centrifugation, the absorbance of the supernatant was measured at 590 nm. One unit of β-glucanase activity was expressed as the amount of enzyme that produces a reducing sugar corresponding to 1 μmol of glucose per minute under the reaction conditions of 40 ° C. and 10 minutes.

  Xylanase activity was quantified by increasing the absorbance at 540 nm by reacting reducing sugar produced by enzymatic hydrolysis using oat spelts-derived xylan as a substrate with DNS. More specifically, 1% xylan substrate solution [Sigma Xylan, from oat spelts dissolved in 200 mM acetate buffer (pH 4.5)] 0.1 mL of culture solution was added to 1.9 mL, and exactly 10 at 40 ° C. After performing the enzyme reaction for 4 minutes, add 4 mL of DNS reagent (including 0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, 22.5% sodium potassium tartrate tetrahydrate, 0.3% lactose monohydrate) and mix well. The reaction was stopped. In order to quantify the amount of reducing sugar contained in the reaction stop solution, the reaction stop solution was accurately heated in a boiling water bath for 15 minutes. Subsequently, after cooling to room temperature, the amount of reducing sugar corresponding to xylose was quantified by measuring the absorbance at 540 nm. One unit of xylanase activity was expressed as the amount of enzyme that produces reducing sugar corresponding to 1 μmol of xylose per minute under the reaction conditions of 40 ° C. and 10 minutes.

Reference Example 2 and Examples 1-6
A liquid medium is produced in the same manner as in Reference Example 1 except that the reaction time of the enzyme reaction and the amount of the enzyme added are changed as shown in Table 1, and the concentration of water-soluble saccharides in the liquid medium is measured to culture Trichoderma reesei. And the enzyme activity of the culture solution was measured. Table 1 shows the measurement results of the water-soluble saccharide concentration and enzyme activity.

Reference example 3
A liquid medium was produced in the same manner as in Reference Example 1 except that the amount of plain paper contained in the raw liquid medium was changed to 3 g, and the reaction time of the enzyme reaction and the addition amount of the enzyme were changed as shown in Table 1. The concentration of water-soluble saccharides in the liquid medium was measured, Trichoderma reesei was cultured, and the enzyme activity of the culture solution was measured. Table 1 shows the measurement results of the water-soluble saccharide concentration and enzyme activity.

Comparative Example 1
Trichoderma reesei was cultured in the same manner as in Reference Example 1 except that the raw material liquid medium was used instead of the liquid medium containing the water-soluble saccharide, and the enzyme activity of the culture solution was measured. The measurement results of enzyme activity are shown in Table 1.

Comparative Example 2
Trichoderma reesei was cultured in the same manner as in Reference Example 3 except that the raw material liquid medium was used instead of the liquid medium containing the water-soluble saccharide, and the enzyme activity of the culture solution was measured. The measurement results of enzyme activity are shown in Table 1.

[Table 1]

  From the results in Table 1, it is understood that the production amounts of β-glucanase and xylanase are significantly improved when the concentration of the water-soluble saccharide contained in the liquid medium for culturing Trichoderma reesei is 1% by weight or more.

Claims (5)

A method for producing cellulase comprising a step of culturing Trichoderma using a liquid medium containing a water-soluble saccharide,
The liquid medium containing the water-soluble saccharide is obtained by adding cellulase to a paper-derived pulp that is not subjected to heat treatment or alkali treatment as a carbon source, and a raw liquid medium containing ammonia nitrogen or amino nitrogen as a nitrogen source, and an enzyme reaction Is obtained by generating water-soluble saccharides,
The concentration of the pulp in the raw material liquid medium is 6% W / V or more,
A method for producing cellulase, wherein the concentration of the water-soluble saccharide in a liquid medium containing the water-soluble saccharide is 1% by weight or more.   The method for producing cellulase according to claim 1, wherein the concentration of the ammonia nitrogen or amino nitrogen in the raw material liquid medium is 35 mM or more.   The method for producing cellulase according to claim 1 or 2, wherein the paper is at least one selected from the group consisting of fine paper, reprint paper, copy paper, newspaper and cardboard.   The method for producing cellulase according to any one of claims 1 to 3, wherein the Trichoderma spp. Is Trichoderma reesei.   The method for producing cellulase according to any one of claims 1 to 4, wherein pulp is not added to a liquid medium containing a water-soluble saccharide in the course of culturing.