JP5599991B2 - Process for producing β-glucanase and xylanase using squeezed koji of fruit and liquid medium - Google Patents

Description

  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 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 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 have limitations on materials that can be used as a carbon source. Even if it is an expensive crystalline cellulose or an inexpensive cellulose resource, it can be used before heat treatment or alkali treatment. It is necessary to perform processing, which requires a relatively high cost.

  For example, Patent Document 1 discloses a substrate for cellulase production in which waste paper is steamed in a ferrous sulfate solution and seeded with 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 ingested. 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 raw material 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, fruit squeezes are generated in large quantities in the production process of juice and the like, but are essentially production waste and lack industrial use. Although some are reused as feed and fertilizer, they have not yet consumed all the amount of fruit squeezed. Therefore, a large amount is treated as industrial waste. However, these sputums are very susceptible to spoilage, and if left untreated, they produce a bad odor and become a major source of pollution, and the establishment of a treatment method is expected. For example, Patent Document 5 discloses a method for producing L-arabinose using such fruit pomace.

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

JP 2003-137901 A Japanese Patent Publication No. 5-33984 JP 9-163980 A JP-A-11-113568 JP 2001-286294 A

  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.

  As a result of intensive studies on the production method of an enzyme for producing β-glucanase and xylanase at the same time and decomposing (saccharifying) the cellulose raw material and producing the enzyme, Useful for producing high-quality β-glucanase and xylanase at the same time by culturing microorganisms belonging to the genus Trichoderma using a) squeezed strawberry and (b) ammonia nitrogen or amino nitrogen Liquid medium was found.

The present invention includes a step of culturing a microorganism belonging to the genus Trichoderma using a liquid medium containing (a) fruit pomace as a carbon source and (b) ammonia nitrogen or amino nitrogen as a nitrogen source. A method for producing glucanase and xylanase comprising :
The concentration of the fruit pomace in the liquid medium is 4-10% W / V,
Provided is a method for producing β-glucanase and xylanase, wherein the concentration of ammonia nitrogen or amino nitrogen in the liquid medium is 40 to 580 mM .

  In one certain form, the microorganisms which belong to the said Trichoderma genus are Trichoderma reesei.

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

  In one certain form, the fruit pomace is an apple pomace.

In addition, the present invention is a liquid medium containing (a) fruit pomace 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. It is,
The concentration of the fruit squeezes is 4-10% W / V,
A liquid medium having a concentration of ammonia nitrogen or amino nitrogen of 40 to 580 mM is provided.

  The present invention contributes to the solution of environmental problems because it effectively uses fruit squeezes to reduce industrial waste. Fruit squeezes are superior in hygienic quality because quality inspection and production process control are strictly conducted at the raw material stage. In addition, since β-glucanase and xylanase, which are cellulolytic enzymes, are simultaneously produced at a high level, it is 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 a culture medium of apple pomace 4%. 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 a culture medium of apple pomace 4%. 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 a culture medium of apple pomace 4%. 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 a culture medium of apple pomace 4%. It is a graph showing the change of the enzyme activity of the culture supernatant liquid with respect to the density | concentration of apple pomace in the culture medium of ammonium sulfate 480 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 a culture medium of apple pomace 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 a culture medium of apple pomace 4%. The concentration of glucose produced when rice straw was saccharified using the supernatants of 4% apple pomace obtained in Example 1 and 1% crystalline cellulose obtained in Reference Example 2, respectively. It is the graph which compared. The concentration of glucose produced when beer cake was saccharified using the respective supernatants of the 4% apple pomace obtained in Example 1 and the 1% crystalline cellulose medium obtained in Reference Example 2. It is the graph which 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 based on a liquid medium (generally referred to as Mandel medium) in which the following medium composition is dissolved and suspended in 100 ml of water. Generally, water is used as a medium, fruit squeezed rice cake as a carbon source, and nitrogen. The source contains ammonia nitrogen or amino nitrogen. An example of a preferred medium composition is shown below.

Medium composition:
Fruit pomace: 4 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 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, 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, fruit pomace is a by-product produced in the production process of juice and the like, and is a residue obtained by squeezing the fruit and then removing the fruit juice by filtration. Fruit squeezes are produced in large quantities in the manufacturing process of juices and are easy to obtain. And since the fruit koji is a by-product of food production, quality inspection and production process management at the raw material stage are strictly carried out, so that hygiene quality is excellent and safe. The fruit pomace is preferably a rosaceae fruit pomace such as apples, pears, peaches, cherries, strawberries and the like. Among these, apple pomace is preferable because the desired enzyme is produced at a high rate.

  The apple varieties may be any apples that have been conventionally used to produce apple juice. For example, “Fuji”, “Tsugaru”, “Wang Lin”, “Jonagar”, “Star King Delicious”, “Miku” Etc. The apple fruit may be ripe or unripe.

  In the manufacturing method of fruit squeezed straw, first, the fruit is washed. At this time, if there are unsuitable raw materials, they are removed. The washed fruit is sent to a crusher and crushed. The crushed fruit is sent to a hydraulic juicer with a pump or the like and squeezed. The squeezed rice cake is then recovered from the juicer. Fruit squeezed rice cake is subjected to treatments such as washing, dehydration, and drying as needed.

  The fruit pomace may be pretreated when it is introduced into the liquid medium. A preferred pretreatment is, for example, delignification treatment. This is because, when lignin is removed from the squeezed buds of fruits, the strong cell wall is broken, cellulose can be easily used, and enzymes are easily produced.

  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.

  Moreover, you may further perform the pretreatment normally performed with respect to the raw material of liquid culture media, such as heat sterilization.

  The concentration of the fruit pomace in the liquid medium is preferably 2% W / V or more. If the concentration of the fruit squeezed koji is less than 2% W / V, the production amount of cellulase, particularly β-glucanase may not increase so much. More preferably, the concentration of the fruit pomace in the liquid medium is 3% W / V or more, more preferably 5% W / V or more.

  The higher the concentration of the fruit pomace in the liquid medium, the better. That is, the upper limit is the limit amount that can stir the liquid medium. 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 fruit pomace concentration in the liquid medium can be 20, 15, 10 or 8% W / V depending on the performance of the agitator. In general, the preferred range of the concentration is 2-15% 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, 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 30 to 660 mM in terms of moles of ammonium. Preferably, it is 40-580 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. Further, the concentration of ammonia nitrogen or amino nitrogen in the liquid medium is preferably increased or decreased according to the concentration of the fruit pomace in the liquid medium, for example, the concentration of the fruit pomace is 4% W / In the case of V, 50 mM is preferable 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. Particularly preferred is Trichoderma reesei.

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

  A normal aeration and agitation culture apparatus is used for liquid culture, and culture is performed 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 using the liquid medium of the present invention. To do. 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, as the cultivation progresses, the fruit pomace in the medium is decomposed, so that supplementing the carbon source may improve the production efficiency of cellulase. The carbon source to be added is not particularly limited as long as it is a material containing natural cellulose and substantially free of nutrients such as starch. Preferred carbon sources to add are fruit squeezed straw, paper, beer straw, barley tea extract straw and the like. Preferred are fruit squeezes, especially the same type of fruit squeezed as originally used as a carbon source for liquid media, since the culture conditions are stabilized.

  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 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 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 ("Xylan, from oat spelts" manufactured by Sigma Co., dissolved in 200 nM acetate buffer (pH 4.5)) was added to 1.9 ml of culture solution or culture supernatant solution 0.1 ml. And an enzyme reaction at 40 ° C. for exactly 10 minutes, followed by DNS reagent (0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, 22.5% potassium potassium tartrate tetrahydrate, 0.3% Add 4 ml (including% 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 is Applicable to the method.

  When cultured, the nutrients in the liquid medium are reduced due to 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, especially when the carbon source is highly contained from the beginning of the culture as described above, the upper limit of the concentration of the fruit pomace is restricted to some extent in consideration of the convenience when the liquid medium is stirred and mixed as described above. It is preferable to do.

  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 Resource Decomposition or Saccharification Method The 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 this 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, wheat 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-mentioned culture solution or culture supernatant solution exhibiting 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. Generally, 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, but at normal pressure and in acetic acid or phosphate buffer, the temperature is 50-60. It is preferable to carry out in the range of 0 degreeC and pH 4-6. 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.

Example 1
Apple fruit (variety "Fuji") was pulverized using a crusher (Amos Co., Ltd. "Hammer Mill"), and subsequently squeezed using an apple juicer (Tsukishima-Andritz "Press Roll Filter"). . Squeeze cake was collected from the juicer, washed with water and dried.

  The apple pomace obtained was removed from the lignin by autoclaving at 121 ° C for 15 minutes in a 0.3N sodium hydroxide aqueous solution, washed thoroughly with water, dried, and pulverized to make the size uniform. Used.

  Trichoderma reesei QM9414 (NBRC 31329) was cultured on potato dextrose agar at 28 ° C. for 7 days to sufficiently form spores. The crystalline cellulose that is the carbon source of Mandel's medium is replaced with 4% (4 g / 100 mL) of apple pomace that has been delignified, and the molar concentration of ammonium nitrogen that is the nitrogen source is 15 mM, 35 mM, 50 mM, and 65 mM, respectively. 100 mM liquid medium adjusted to pH 4.8 with phosphoric acid or sodium hydroxide was prepared in a 500 mL baffle 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 determined 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. % 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 a reducing sugar produced by enzymatic hydrolysis using “oat 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)] 0.1 mL of culture solution was added to 1.9 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 that is the carbon source of the Mandel medium is replaced with delignified apple pomace 4% (4 g / 100 mL) obtained in the same manner as in Example 1, and the ammonium sulfate that is the nitrogen source is replaced with ammonium chloride to give ammonia. A liquid medium was prepared in the same manner as in Example 1 by adding 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 apple pomace 4% (4 g / 100 mL) obtained in the same manner as in Example 1, and the ammonium sulfate as the nitrogen source was replaced with leucine. A liquid medium was prepared in the same manner as in Example 1 by adding so that the molar concentration of nitrogen was 8 mM, 15 mM, 23 mM, 31 mM, 38 mM, 46 mM, or 53 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 4
The crystalline cellulose as the carbon source of the Mandel medium was replaced with delignified apple pomace 4% (4 g / 100 mL) obtained in the same manner as in Example 1, and the ammonium source as the nitrogen source was replaced with urea to form an amino form. A liquid medium was prepared in the same manner as in Example 1 by adding so that the molar concentration of nitrogen was 17 mM, 33 mM, 50 mM, 67 mM, 83 mM, or 100 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 5
The crystalline cellulose that is the carbon source of the Mandel medium is replaced with delignified apple pomace obtained in the same manner as in Example 1, and the concentration is 1%, 2%, 3%, 4%, 5%, 6%, or 7 A liquid medium was prepared in the same manner as in Example 1 by adding so that the molar concentration of ammonium nitrogen of ammonium sulfate as a nitrogen source was 480 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 Mandel medium, is set to 1%, and the ammonia nitrogen, which is the nitrogen source, is added so that the molar concentration of ammonium 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, which 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, is added in place of delignified apple pomace 1% (3 g / 100 mL) obtained in the same manner as in Example 1, and is also added as the nitrogen source. Were added so that the molar concentration of each was 15 mM, 35 mM, 50 mM, 65 mM, 80 mM, 100 mM, or 115 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 as the carbon source of the Mandel medium was replaced with delignified apple pomace 4% (3 g / 100 mL) obtained in the same manner as in Example 1, and the molar concentration of ammonium nitrogen in the ammonium sulfate as the nitrogen source. Were added so as to be 330 mM, 420 mM, 500 mM, 580 mM, 660 mM, 720 mM or 800 mM, respectively, to prepare a liquid medium 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 7
The culture supernatant obtained in Example 1 (4% apple pomace, ammonia nitrogen is 100 mM ammonium sulfate) and the culture supernatant obtained in Reference Example 2 (1% crystalline cellulose, ammonia nitrogen is 100 mM) A cellulose saccharification test was conducted using ammonium sulfate. The cellulose raw material used for saccharification was delignified by the following method. Rice straw and beer straw were each 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 liquid: 9.5 mL, a 1 M acetic acid buffer (pH 4.8): 0.2 mL (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. 10 and FIG.

  Β-glucanase and xylanase which 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 for producing ethanol from cellulose resources.

Claims (5)

Β-glucanase and xylanase comprising a step of culturing a microorganism belonging to the genus Trichoderma using (a) a fruit pomace as a carbon source and (b) a liquid medium containing ammonia nitrogen or amino nitrogen as a nitrogen source A manufacturing method of
The concentration of the fruit pomace in the liquid medium is 4-10% W / V,
A method for producing β-glucanase and xylanase, wherein the concentration of ammonia nitrogen or amino nitrogen in the liquid medium is 40 to 580 mM . The method for producing β-glucanase and xylanase according to claim 1 , wherein the microorganism belonging to the genus Trichoderma is Trichoderma reesei. The method for producing β-glucanase and xylanase according to claim 1 or 2 , wherein fruit pomace is added to the liquid medium in the course of culturing. The method for producing β-glucanase and xylanase according to any one of claims 1 to 3 , wherein the fruit pomace is apple pomace. (A) a fruit culture as a carbon source, and (b) a liquid medium containing ammonia nitrogen or amino nitrogen as a nitrogen source, which is used for culturing microorganisms belonging to the genus Trichoderma ,
The concentration of the fruit squeezes is 4-10% W / V,
A liquid medium in which the concentration of ammonia nitrogen or amino nitrogen is 40 to 580 mM .

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