WO2010116545A1

WO2010116545A1 - METHOD FOR PRODUCING β-GLUCANASE AND XYLANASE USING SPENT GRAINS AND LIQUID MEDIUM

Info

Publication number: WO2010116545A1
Application number: PCT/JP2009/064176
Authority: WO
Inventors: 和郎福田
Original assignee: アサヒビール株式会社
Priority date: 2009-04-10
Filing date: 2009-08-11
Publication date: 2010-10-14
Also published as: JPWO2010116545A1

Abstract

To produce a cellulase, which has an excellent ability to degrade a cellulose resource containing xylan, at a low cost. A method for producing β-glucanase and xylanase which comprises a step of culturing a microorganism belonging to the genus Trichoderma with the use of a liquid medium containing (a) spent grains as a carbon source and (b) ammonia nitrogen or amino nitrogen as a nitrogen source.

Description

Process for producing β-glucanase and xylanase using beer lees and liquid medium

The present invention relates to a production method for producing β-glucanase and xylanase at the same time and a liquid medium useful for producing the enzyme.

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 cellooligosaccharide and cellobiose by its action. Cellulolytic enzymes are generally called cellulases.

When attempting to artificially produce 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 using a medium containing nutrients such as a carbon source and a nitrogen source to secrete cellulase is also known.

However, the conventional methods for producing cellulase are limited in materials and pretreatment methods that can be used as a carbon source. Even if it is expensive crystalline cellulose or an inexpensive cellulose resource, heat treatment or alkali It is necessary to perform preprocessing such as processing, which requires a relatively high cost.

For example, Patent Document 1 discloses a cellulase production substrate capable of steaming used paper in a ferrous sulfate solution and inoculating cellulase producing bacteria. Patent Document 2 discloses a method for producing a substrate for cellulase production in which finely pulverized bagasse is cooked with caustic, treated with a hypochlorite solution, and a cellulase-producing bacterium, Trichoderma reesei, can be inoculated. is doing.

Cellulases obtained by these conventional methods mainly contain β-glucanase, have low xylanase activity, and are inferior in decomposing ability of cellulose resources containing xylan such as bagasse and rice straw. Therefore, the effect is low for the purpose of effective utilization of various naturally occurring cellulose resources.

Patent Document 3 discloses a method for producing cellulase, which includes a step of subjecting a mutant strain belonging to Trichoderma reesei to liquid culture and collecting the obtained cellulase. As the carbon source of the medium, various materials having different chemical structures and properties such as cellulose powder, cellobiose, filter paper, general paper, sawdust, bran, rice bran, bagasse, soybean meal, and coffee meal are listed (No. 0011). Paragraph).

However, only cellobiose (Example 1) and Avicel (Example 2) are actually used in the culturing operation, and the production of cellulase has been confirmed for other materials, that is, natural cellulose materials. Absent.

Patent Document 4 manufactures xylanase by culturing microorganisms belonging to the genus Trichoderma using dilute alcohol distillate waste liquor that has been subjected to pretreatment such as removal of solid components, concentration of non-volatile components, and autoclaving of the concentrate. The method of doing is disclosed.

However, rye used as a carbon source in this technology is difficult to obtain and requires a high cost because it requires a more complicated pretreatment, and in this method, the amount of β-glucanase produced decreases. End up.

On the other hand, beer lees are generated in large quantities in the beer manufacturing process, but are essentially production waste and lack industrial use. Although it is partially reused as feed, it has not yet consumed all the amount of beer lees. Therefore, a large amount is treated as industrial waste. However, beer lees are very susceptible to spoilage, and if left untreated, a bad odor is generated, which is a major source of pollution, and the establishment of a treatment method is expected. For example, patent document 5 is disclosing the method of manufacturing a good-quality compost in a short time using this beer lees.

However, it has not been known so far to use beer lees as a medium for high production of β-glucanase and xylanase simultaneously.

JP 2003-137901 A Japanese Patent Publication No. 5-33984 JP-A-9-163980 JP-A-11-113568 JP-A-8-26871

The present invention solves the above-mentioned conventional problems, and an object of the present invention is to produce a cellulase excellent in decomposing ability of cellulose resources containing xylan at a low cost.

The inventors of the present invention have made extensive investigations on a method for producing an enzyme that simultaneously produces high production of β-glucanase and xylanase and decomposes (saccharifies) a cellulose raw material, and the production of the enzyme. A production method for simultaneously producing β-glucanase and xylanase by culturing a microorganism belonging to the genus Trichoderma using beer lees and (b) ammonia nitrogen or amino nitrogen, and a liquid useful for producing the enzyme The medium was found.

The present invention provides a β-glucanase comprising the step of culturing a microorganism belonging to the genus Trichoderma using (a) a beer lees as a carbon source and (b) a liquid medium containing ammonia nitrogen or amino nitrogen as a nitrogen source. A method for producing xylanase is provided.

In one embodiment, the concentration of the beer cake in the liquid medium is 2% W / V or more.

In one embodiment, the concentration of the beer cake in the liquid medium is 3 to 15% W / V.

In one embodiment, the concentration of the ammonia nitrogen or amino nitrogen in the liquid medium is 30 to 660 mM.

In one embodiment, the concentration of the ammonia nitrogen or amino nitrogen in the liquid medium is 40 to 580 mM.

In one embodiment, the microorganism belonging to the genus Trichoderma is Trichoderma reesei.

In one certain form, a carbon source is added with respect to the said liquid culture medium in the process of culture | cultivation.

The present invention also relates to a liquid medium containing (a) beer lees as a carbon source and (b) ammonia nitrogen or amino nitrogen as a nitrogen source, which is used for culturing microorganisms belonging to the genus Trichoderma. I will provide a.

In one embodiment, the beer lees are contained at 2% W / V or more.

In one embodiment, 30 to 660 mM of ammonia nitrogen or amino nitrogen is contained.

The present invention also provides β-glucanase and xylanase produced by any of the methods described above.

The present invention also provides a method for decomposing or saccharifying cellulose resources, characterized by using the β-glucanase and xylanase.

The present invention contributes to the solution of environmental problems because it effectively uses beer lees to reduce industrial waste. Beer lees are superior in hygiene quality because quality inspection and production process management at the raw material stage are strictly conducted. In addition, since β-glucanase and xylanase, which are cellulolytic enzymes, are simultaneously produced at a high level, they are extremely useful for saccharification of natural cellulose resources such as bagasse and rice straw. In particular, it is useful for biomass ethanol production in which ethanol is produced from cellulose resources.

It is a graph showing the change of the enzyme activity of the culture supernatant liquid with respect to the density | concentration of ammonium sulfate in the culture medium of 3% of beer lees. It is a graph showing the change of the enzyme activity of the culture supernatant liquid with respect to the density | concentration of ammonium chloride in the culture medium of 3% of beer lees. It is a graph showing the change of the enzyme activity of the culture supernatant liquid with respect to the density | concentration of leucine in the culture medium of 3% of beer lees. It is a graph showing the change of the enzyme activity of the culture supernatant liquid with respect to the density | concentration of ammonia in the culture medium of beer lees 3%. It is a graph showing the change of the enzyme activity of the culture supernatant liquid with respect to the density | concentration of urea in the culture medium of beer lees 3%. It is a graph showing the change of the enzyme activity of the culture supernatant liquid with respect to the density | concentration of beer lees in the culture medium of ammonium sulfate 320 mM. It is a graph showing the change of the enzyme activity of the culture supernatant liquid with respect to the density | concentration of ammonium sulfate in the culture medium of crystalline cellulose 1%. It is a graph showing the change of the enzyme activity of the culture supernatant liquid with respect to the density | concentration of a crystalline cellulose in the culture medium of ammonium sulfate 160 mM. It is a graph showing the change of the enzyme activity of the culture supernatant liquid with respect to the density | concentration of ammonium sulfate in the culture medium of beer lees 1%. It is a graph showing the change of the enzyme activity of the culture supernatant liquid with respect to the density | concentration of ammonium sulfate in the culture medium of 3% of beer lees. When the rice straw was saccharified using the supernatant of the beer lees 3% obtained in Example 1 and the crystalline cellulose 1% obtained in Reference Example 2, the concentration of the produced glucose was determined. It is the graph compared. When beer koji was saccharified using the supernatants of the beer koji 3% medium obtained in Example 1 and the crystalline cellulose 1% medium obtained in Reference Example 2, the concentration of the produced glucose was determined. It is the graph compared.

Liquid medium The liquid medium of the present invention is a microorganism that produces a cellulolytic enzyme, such as Trichoderma, Aspergillus, Acremonium, Sporotricum, Penicillium, Tallomyces, Humicola, Neocalimasticus, Thermomyces, or Cross It contains nutrients for growing microorganisms belonging to the genus Tridium and Streptomyces.

Such a liquid medium is prepared on the basis of a liquid medium (generally called Mandel medium) in which the following medium composition is dissolved and suspended in 100 ml of water. Generally, water is used as a medium, beer lees as a carbon source, and nitrogen source. It contains ammonia nitrogen or amino nitrogen. An example of a preferred medium composition is shown below.

Medium composition:
Beer lees: 3 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 element solution (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, 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 the present invention, beer cake is a by-product in the beer production process, and is a residue obtained by saccharifying malt from which barley has been germinated and then filtering off wort. It is not restricted to the type of barley, the type of auxiliary material, and the like, and the residue produced as a by-product in the production process such as happoshu with reduced malt use ratio is also included in the beer lees of the present invention.

Beer lees are generated in large quantities in the beer manufacturing process and are easy to obtain. And since beer lees are a by-product of food production, quality inspection and production process management at the raw material stage are strictly performed, so that hygiene quality is excellent and safe. Examples of types of beer lees include raw beer lees, dehydrated beer lees, and dried beer lees.

The beer lees may be pretreated when introduced into the liquid medium. Preferred pretreatments are, for example, grinding treatment and delignification treatment. This is because when lignin is removed from beer lees, the strong cell wall is broken, cellulose can be easily used, and enzymes are easily produced. Moreover, delignification treatment can be performed more efficiently by pulverizing beer lees.

The method of delignification treatment is not particularly limited, for example, a method of heating and decomposing in the presence of a strong alkaline substance such as sodium hydroxide or a strong acidic substance such as sulfuric acid or phosphoric acid, Examples thereof include a method of decomposing by microorganisms and a method of decomposing by hydrothermal treatment under high temperature and high pressure. Considering the load on the treatment equipment and the environment, a method of decomposing by hydrothermal treatment at high temperature and high pressure is preferable.

Further, a pretreatment usually performed on the raw material of the liquid medium such as heat sterilization may be further performed.

It is preferable that the concentration of beer lees in the liquid medium is 2% W / V or more. When the concentration of beer lees is less than 2% W / V, the production amount of cellulase, particularly β-glucanase may not increase so much. More preferably, the concentration of beer lees in the liquid medium is 3% W / V or more, more preferably 4% W / V or more, 5% W / V or more, 6% W / V or more, or 7% W / V or more. It is.

The higher the concentration of beer lees in the liquid medium, the better. That is, the upper limit is an amount that allows the liquid medium to be stirred and mixed. This is because if the liquid medium cannot be stirred, the microorganisms are not mixed uniformly in the liquid medium, and the culture does not proceed normally. The upper limit of the concentration of beer lees in the liquid medium can be 20, 15, 10 or 8% W / V depending on the performance of the stirrer. In general, the preferred range of the concentration is 3-15% W / V, preferably 4-10% W / V.

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, leucine, sodium glutamate).

Of these, ammonium sulfate is a particularly preferred compound for use in the liquid medium of the present invention as a nitrogen source. 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 30 to 660 mM in terms of moles of ammonium. Preferably, it is 40 to 580 mM. When the concentration is less than 30 mM, the production amount of cellulase, particularly β-glucanase may not increase so much. On the other hand, when the 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 beer lees in a liquid medium, for example, when the concentration of beer lees is 3% W / V Is preferably 50 mM in consideration of cost and the like.

Production method of β-glucanase and xylanase The microorganism belonging to the genus Trichoderma used in the present invention is not particularly limited as long as it produces cellulase necessary for saccharification of cellulose. A preferred microorganism belonging to the genus Trichoderma is a Trichoderma filamentous fungus, specifically, Trichoderma reesei or Trichoderma viride. Trichoderma reesei is particularly preferable from the viewpoint of high production of β-glucanase and xylanase at the same time.

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

In the liquid culture, a normal aeration and agitation culture apparatus is used, and the above liquid medium is used for culturing at a culture temperature of 20 to 33 ° C., preferably 28 to 30 ° C. and a culture pH of 4 to 6 for 4 to 10 days. When the liquid medium is used from the beginning of the culture, the concentration of components (for example, carbon source and nitrogen source) contained in the liquid medium corresponds to the initial concentration of the components in the culture method of the present invention.

A carbon source may be added to the liquid medium during the culture process. This is because beer lees in the medium are decomposed as the culture progresses, so that supplementation with a carbon source may improve cellulase production efficiency. The carbon source to be added is preferably a material containing natural cellulose and low in nutrients such as starch. Preferred carbon sources to add are beer lees, barley tea extract lees, paper, fruit pomace (especially apple pomace), wheat bran and the like.

When adding a carbon source, the additional form may be a continuous type or a batch type, and the additional time and amount may be adjusted so that stirring and mixing are possible even after the addition.

In the case of adding a carbon source, ammonia nitrogen or amino nitrogen may be added as needed during the culture process.

Then, if necessary, the cells are removed from the culture solution by a known method such as centrifugation or filtration to obtain a Trichoderma filamentous fungus culture supernatant. The Trichoderma filamentous fungus 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 30 U / mL or more, preferably 50 U / mL or more, more preferably 60 U / mL or more, and further preferably 70 U / mL or more. Moreover, the xylanase activity of this culture solution or culture supernatant is 25 U / mL or more, preferably 30 U / mL or more, more preferably 40 U / mL or more, and further preferably 50 U / mL or more. When either β-glucanase activity or xylanase activity in the culture solution or culture supernatant falls below the lower limit, the effect on the purpose of effective utilization of various naturally occurring cellulose resources is reduced.

The hemicellulase activity can be quantified by increasing the absorbance at 540 nm by reacting a reducing sugar produced by enzymatic hydrolysis using xylan derived from “oat spelts” as a substrate with DNS.

More specifically, 1% xylan substrate solution (Sigma's “Xylan, from oat spelts” dissolved in 200 nM acetate buffer (pH 4.5)) was added to 1.9 mL of culture solution or culture supernatant 0.1 mL. Then, after the enzyme reaction was carried out at 40 ° C. for exactly 10 minutes, DNS reagent (0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, 22.5% sodium potassium tartrate tetrahydrate, 0. Add 4 mL (including 3% lactose 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 hemicellulase 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.

In the present invention, “culturing a microorganism belonging to the genus Trichoderma” refers to an operation for growing the microorganism according to common general technical knowledge. That is, in the method of performing liquid culture for the purpose of producing β-glucanase and xylanase, if there is a process in which a microorganism belonging to the genus Trichoderma exists in at least the liquid medium of the present invention, the culture method of the present invention can be used. Applicable to the method.

When culture is performed, the nutrients in the liquid medium are reduced by consumption by microorganisms belonging to the genus Trichoderma. Therefore, at the end of the culture, the concentration of the carbon source or nitrogen source in the medium becomes less than the predetermined concentration, and as a result, a microorganism belonging to the genus Trichoderma may grow in a medium not corresponding to the liquid medium of the present invention. unknown. Even in such a case, for example, when the liquid medium to be used corresponds to the liquid medium of the present invention containing a carbon source or a nitrogen source at a predetermined concentration at the start of the culture, the present invention is at least in the initial stage of the culture. Since the microorganism belonging to the genus Trichoderma grows in the liquid medium, the culture method naturally corresponds to the method of the present invention.

In addition, in the case where the carbon source is contained in a high amount from the start of the culture in this way, as described above, the upper limit of the concentration of the beer koji is limited to some extent in consideration of the convenience when the liquid medium is stirred and mixed. Is preferred.

On the other hand, even if the culture is performed using a medium that does not correspond to the liquid medium of the present invention, the concentration of the carbon source or nitrogen source in the medium is lower than the predetermined concentration at the initial stage of the culture. When the concentration of the carbon source or nitrogen source in the medium becomes a predetermined concentration or more after that, the microorganism belonging to the genus Trichoderma grows in the liquid medium of the present invention. This corresponds to the method of the present invention.

Cellulose Resources Decomposition or Saccharification Method β-glucanase and xylanase obtained by the method of the present invention are 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 where the enzyme is not inactivated. In general, when the reaction is performed at normal pressure, the temperature is 30 to 70 ° C., and the pH is 3 to 7. Range may be sufficient. The pressure, temperature and pH are also adjusted as appropriate so that glucose can be obtained in a higher yield, as described above. However, the temperature is 50-60 in acetic acid or phosphate buffer at normal pressure. It is preferably carried out in the range of 4 ° C. and pH 4-6. The reaction time is generally 6 to 147 hours, preferably 24 to 72 hours.

An aqueous solution containing glucose is obtained by saccharification of cellulose. 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 treatment, 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.

Hereinafter, the present invention will be described more specifically by way of examples. However, the present invention is not limited to these examples.

Example 1
In the process of producing beer, malt germinated from barley was saccharified, and wort was filtered to collect a residue. This residue was washed with water and dried to obtain a beer lees. The obtained beer koji was hydrothermally treated at 121 ° C., 2 bar, 15 minutes in a 0.3N aqueous sodium hydroxide solution to remove lignin, washed thoroughly with water, and then dried.

Trichoderma reesei QM9414 (NBRC 31329) was cultured on a potato dextrose agar medium at 28 ° C. for 7 days to sufficiently form spores. The crystalline cellulose that is the carbon source of the Mandel medium is replaced with delignified 3% (3 g / 100 mL) of beer lees, and the molar concentration of ammonium nitrogen that is the nitrogen source is 15 mM, 35 mM, 50 mM, 65 mM, A 100 mM liquid medium added to 80 mM, 100 mM or 115 mM and adjusted to pH 4.8 with phosphoric acid or sodium hydroxide was prepared in a 500 mL baffled Erlenmeyer flask. One platinum loop of the cultured Trichoderma reesei was taken into this liquid medium and cultured with shaking at 28 ° C., 180 rpm for 7 days. On the seventh day, the culture solution was centrifuged, and β-glucanase activity and xylanase activity of the supernatant were measured.

(Measurement of enzyme activity)
The enzyme activity of the culture solution obtained above was measured.
The β-glucanase activity was measured by measuring the absorbance of a stained fragment produced by enzymatic degradation using a dye-labeled β-glucan as 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 accurately at 40 ° C. for 10 minutes, and then the stop solution [4% sodium acetate, 0.4 % Zinc acetate and 80% methyl cellosolve (pH 5)] 0.6 mL 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.

Next, the xylanase activity was quantified by increasing the absorbance at 540 nm by reacting the reducing sugar produced by enzymatic hydrolysis using “oat の spelts” -derived xylan as a substrate with DNS. More specifically, 1% xylan substrate solution [Sigma's “Xylan, from oat spelts” dissolved in 200 mM acetate buffer (pH 4.5)] was added to 1.9 mL of the culture solution 0.1 mL, and the mixture was heated to 40 ° C. After an enzyme reaction for exactly 10 minutes, a DNS reagent (0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, 22.5% sodium potassium tartrate tetrahydrate, 0.3% lactose / water) 4 mL (including Japanese product) was added and mixed well to stop the reaction. 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 a reducing sugar corresponding to 1 μmol of xylose per minute under the reaction conditions of 40 ° C. and 10 minutes. The results are shown in FIG.

Example 2
The crystalline cellulose as the carbon source of the Mandel medium was replaced with delignified 3% (3 g / 100 mL) of delignified beer koji obtained in the same manner as in Example 1, and the ammonium sulfate as the nitrogen source was replaced with ammonium chloride. A liquid medium was prepared in the same manner as in Example 1 by adding nitrogen so that the molar concentration of nitrogen was 20 mM, 40 mM, 50 mM, 60 mM, 80 mM, 100 mM, or 120 mM. Trichoderma reesei QM9414 (NBRC 31329) is cultured on potato dextrose agar medium at 28 ° C. for 7 days to fully form spores. This platinum loop is inoculated into a liquid medium and shaken at 28 ° C., 180 rpm for 7 days. Cultured. On day 7, the culture broth was centrifuged, and β-glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.

Example 3
The crystalline cellulose as the carbon source of the Mandel medium was replaced with delignified 3% (3 g / 100 mL) of delignified beer obtained as in Example 1, and the ammonium sulfate as the nitrogen source was replaced with leucine, resulting in amino nitrogen Was added so that the molar concentration of each was 8 mM, 15 mM, 23 mM, 31 mM, 38 mM, 46 mM, or 53 mM, and a liquid medium was prepared in the same manner as in Example 1. Trichoderma reesei QM9414 (NBRC 31329) is cultured on potato dextrose agar medium at 28 ° C. for 7 days to fully form spores. This platinum loop is inoculated into a liquid medium and shaken at 28 ° C., 180 rpm for 7 days. Cultured. On day 7, the culture broth was centrifuged, and β-glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.

Example 4
The crystalline cellulose as the carbon source of the Mandel medium was replaced with delignified beer lees 3% (3 g / 100 mL) obtained in the same manner as in Example 1, and the ammonium sulfate as the nitrogen source was replaced with aqueous ammonia to give a molar concentration. Were added so as to be 15 mM, 30 mM, 45 mM, 60 mM, 75 mM, 90 mM or 105 mM, respectively, to prepare a liquid medium in the same manner as in Example 1. Trichoderma reesei QM9414 (NBRC 31329) is cultured on potato dextrose agar medium at 28 ° C. for 7 days to fully form spores. This platinum loop is inoculated into a liquid medium and shaken at 28 ° C., 180 rpm for 7 days. Cultured. On day 7, the culture broth was centrifuged, and β-glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.

Example 5
The crystalline cellulose that is the carbon source of the Mandel medium was replaced with delignified 3% (3 g / 100 mL) of delignified beer obtained as in Example 1, and the ammonium sulfate that was the nitrogen source was replaced with urea to form amino nitrogen Was added so that the molar concentration of each was 17 mM, 33 mM, 50 mM, 67 mM, 83 mM, or 100 mM, and a liquid medium was prepared in the same manner as in Example 1. Trichoderma reesei QM9414 (NBRC 31329) is cultured on potato dextrose agar medium at 28 ° C. for 7 days to fully form spores. This platinum loop is inoculated into a liquid medium and shaken at 28 ° C., 180 rpm for 7 days. Cultured. On day 7, the culture broth was centrifuged, and β-glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.

Example 6
The crystalline cellulose that is the carbon source of the Mandel medium is replaced with delignified beer koji obtained in the same manner as in Example 1 so that the concentration becomes 1%, 2%, 3%, 4%, 5%, or 6%. In addition, a liquid medium was prepared in the same manner as in Example 1 by adding ammonium nitrate as the nitrogen source so that the molar concentration of ammonia nitrogen was 320 mM. Trichoderma reesei QM9414 (NBRC 31329) is cultured on potato dextrose agar medium at 28 ° C. for 7 days to fully form spores. This platinum loop is inoculated into a liquid medium and shaken at 28 ° C., 180 rpm for 7 days. Cultured. On day 7, the culture broth was centrifuged, and β-glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.

Reference example 1
The concentration of crystalline cellulose, which is the carbon source of the Mandel medium, is set to 1%, and the ammonium nitrate ammonium nitrogen, which is the nitrogen source, is added so that the molar concentration of ammonia nitrogen is 15 mM, 35 mM, 50 mM, 65 mM, 80 mM, 100 mM, or 115 mM, respectively. A liquid medium was prepared in the same manner as in Example 1. Trichoderma reesei QM9414 (NBRC 31329) is cultured on potato dextrose agar medium at 28 ° C. for 7 days to fully form spores. This platinum loop is inoculated into a liquid medium and shaken at 28 ° C., 180 rpm for 7 days. Cultured. On day 7, the culture broth was centrifuged, and β-glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.

Reference example 2
It is added so that the concentration of crystalline cellulose that is the carbon source of Mandel medium is 1%, 1.5%, 2%, 2.5%, 3%, 3.5% or 4%, and it is also a nitrogen source A liquid medium was prepared in the same manner as in Example 1 by adding ammonium sulfate to a molar concentration of ammonia nitrogen of 160 mM. Trichoderma reesei QM9414 (NBRC 31329) is cultured on potato dextrose agar medium at 28 ° C. for 7 days to fully form spores. This platinum loop is inoculated into a liquid medium and shaken at 28 ° C., 180 rpm for 7 days. Cultured. On day 7, the culture broth was centrifuged, and β-glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.

Reference example 3
The crystalline cellulose, which is the carbon source of the Mandel medium, was added in place of delignified beer lees 1% (3 g / 100 mL) obtained in the same manner as in Example 1, and the ammonium nitrogen of ammonium sulfate as the nitrogen source was added. A liquid medium was prepared in the same manner as in Example 1 by adding the molar concentrations to 15 mM, 35 mM, 50 mM, 65 mM, 80 mM, 100 mM, and 115 mM, respectively. Trichoderma reesei QM9414 (NBRC 31329) is cultured on potato dextrose agar medium at 28 ° C. for 7 days to fully form spores. This platinum loop is inoculated into a liquid medium and shaken at 28 ° C., 180 rpm for 7 days. Cultured. On day 7, the culture broth was centrifuged, and β-glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.

Example 7
The crystalline cellulose, which is the carbon source of the Mandel medium, is replaced with delignified 3% (3 g / 100 mL) of delignified beer koji obtained in the same manner as in Example 1, and the ammonium nitrogen molar concentration of ammonium sulfate, which is the nitrogen source, is changed. Liquid media were prepared in the same manner as in Example 1 by adding 330 mM, 420 mM, 500 mM, 580 mM, 660 mM, 720 mM or 800 mM. Trichoderma reesei QM9414 (NBRC 31329) is cultured on potato dextrose agar medium at 28 ° C. for 7 days to fully form spores. This platinum loop is inoculated into a liquid medium and shaken at 28 ° C., 180 rpm for 7 days. Cultured. On day 7, the culture broth was centrifuged, and β-glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.

Example 8
The culture supernatant obtained in Example 1 (3% beer lees, ammonia nitrogen is 100 mM ammonium sulfate) and the culture supernatant obtained in Reference Example 2 (1% crystalline cellulose, ammonium nitrogen is 100 mM ammonium sulfate) ) Was used to conduct a saccharification test on cellulose raw materials. Rice straw and beer straw were prepared as cellulose raw materials for saccharification. These cellulose raw materials were delignified by the following method.

The cellulose raw material was finely pulverized, suspended in 0.3N NaOH, treated at 120 ° C. for 15 minutes, thoroughly washed with water, and dried. The saccharification of the cellulose raw material is carried out by using a cellulose raw material: 0.3 g, a culture supernatant: 9.5 mL, 1M acetic acid buffer (pH 4.8): 0.2 mL of a liquid (cellulose raw material 3% liquid) at 50 ° C., pH 4. The saccharified product was shaken for 8, 48 hours, and the produced glucose was measured with Glucose CII-Test Wako (Wako Pure Chemical Industries). The results are shown in FIG. 11 and FIG.

Β-glucanase and xylanase that are extremely useful for saccharification of natural cellulose resources such as bagasse and rice straw can be produced at the same time, and can be used for biomass ethanol production to produce ethanol from cellulose resources.

Claims

Production of β-glucanase and xylanase comprising a step of culturing a microorganism belonging to the genus Trichoderma using a liquid medium containing (a) beer lees as a carbon source and (b) ammonia nitrogen or amino nitrogen as a nitrogen source Method.
The method for producing β-glucanase and xylanase according to claim 1, wherein the concentration of the beer lees in the liquid medium is 2% W / V or more.
The method for producing β-glucanase and xylanase according to claim 1 or 2, wherein the concentration of the beer koji in the liquid medium is 3 to 15% W / V.
The method for producing β-glucanase and xylanase according to any one of claims 1 to 3, wherein the concentration of the ammonia nitrogen or amino nitrogen in the liquid medium is 30 to 660 mM.
The method for producing β-glucanase and xylanase according to any one of claims 1 to 4, wherein the concentration of the ammonia nitrogen or amino nitrogen in the liquid medium is 40 to 580 mM.
The method for producing β-glucanase and xylanase according to any one of claims 1 to 5, wherein the microorganism belonging to the genus Trichoderma is Trichoderma reesei.
The method for producing β-glucanase and xylanase according to any one of claims 1 to 6, wherein a carbon source is added to the liquid medium in the course of culturing.
(A) A liquid medium containing beer lees as a carbon source and (b) ammonia nitrogen or amino nitrogen as a nitrogen source, which is used for culturing microorganisms belonging to the genus Trichoderma.
The liquid medium according to claim 8, wherein the beer cake contains 2% W / V or more.
The liquid medium according to claim 8 or 9, which contains 30 to 660 mM of ammonia nitrogen or amino nitrogen.
A β-glucanase and xylanase produced by the method according to any one of claims 1 to 7.
A method for decomposing or saccharifying cellulose resources, characterized by using the β-glucanase and xylanase according to claim 11.