WO2010070748A1 - β-グルカナーゼ及びキシラナーゼの製造方法及び液体培地 - Google Patents
β-グルカナーゼ及びキシラナーゼの製造方法及び液体培地 Download PDFInfo
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- WO2010070748A1 WO2010070748A1 PCT/JP2008/072981 JP2008072981W WO2010070748A1 WO 2010070748 A1 WO2010070748 A1 WO 2010070748A1 JP 2008072981 W JP2008072981 W JP 2008072981W WO 2010070748 A1 WO2010070748 A1 WO 2010070748A1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/248—Xylanases
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/244—Endo-1,3(4)-beta-glucanase (3.2.1.6)
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01006—Endo-1,3(4)-beta-glucanase (3.2.1.6)
Definitions
- the present invention relates to a method for producing ⁇ -glucanase and xylanase and a liquid medium.
- cellulose is mainly degraded by microorganisms, and it is known that various microorganisms such as bacteria and filamentous fungi produce cellulose-degrading enzymes.
- Cellulolytic enzymes are generally called cellulases.
- Trichoderma 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.
- 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.
- 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 ⁇ -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 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.
- Non-Patent Document 1 shows that cellulase productivity is low in an enzyme production test by Trichoderma reesei using paper (newspaper or office paper) that is not subjected to pretreatment such as heat treatment or alkali treatment. Has been.
- 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 present invention belongs to the genus Trichoderma, using (a) pulp derived from paper not subjected to heat treatment or alkali treatment as a carbon source, and (b) a liquid medium containing ammonia nitrogen or amino nitrogen as a nitrogen source.
- a method for producing ⁇ -glucanase and xylanase which comprises a step of culturing a microorganism.
- the initial concentration of the pulp in the liquid medium is 2% W / V or more.
- the initial concentration of the pulp in the liquid medium is 2 to 7% W / V.
- the initial concentration of the ammonia nitrogen or amino nitrogen in the liquid medium is 50 mM or more.
- the initial concentration of the ammonia nitrogen or amino nitrogen in the liquid medium is 50 to 660 mM.
- the paper is at least one selected from the group consisting of high-quality paper, reprint paper, copy paper, newspaper and cardboard.
- the microorganism belonging to the genus Trichoderma is Trichoderma reesei.
- pulp is added to the liquid medium in the course of culturing.
- the present invention also relates to (a) a paper-derived pulp that is not subjected to heat treatment or alkali treatment as a carbon source, and (b) a liquid medium containing ammonia nitrogen or amino nitrogen as a nitrogen source, Provided is a liquid medium used for culturing microorganisms belonging to.
- the pulp contains 2% W / V or more.
- the ammonia nitrogen or amino nitrogen is contained in an amount of 50 to 660 mM.
- the present invention also provides ⁇ -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 ⁇ -glucanase and xylanase.
- untreated paper can be used as the carbon source of the liquid medium, the cost is low and the energy is low, and the burden on the environment is small.
- ⁇ -glucanase and xylanase are simultaneously produced at high yields as cellulases, which is extremely useful for saccharification of natural cellulose resources containing xylan such as bagasse and rice straw.
- it is useful for biomass ethanol production in which ethanol is produced from cellulose resources.
- the liquid medium of the present invention is a material containing nutrients for growing microorganisms belonging to the genus Trichoderma.
- a liquid medium is prepared on the basis of a liquid medium (generally referred to as Mandel medium) in which the following medium composition is dissolved and suspended in 100 ml of water, pulp as a carbon source, and ammonia nitrogen or amino nitrogen as a nitrogen source. Is included.
- Mandel medium a liquid medium
- An example of a preferred medium composition is shown below.
- Crystalline cellulose (Fluka BioChemika, trade name Avicel PH101): 1 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: 0.03g, 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 40mg, MnCl 2 ⁇ 4H 2 O 8mg, ZnSO 4 ⁇ 7H 2 O 200mg solution): 0.1 mL, water: including 100 mL (to pH4.8 with phosphoric acid or sodium hydroxide Adjustment)
- Pulp is a fiber used as a raw material for paper production.
- 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.
- preferable papers include high-quality paper, reprint paper, copy paper, newspaper, and cardboard paper.
- the papers only need to contain a preferable pulp, and may be printed or written or generally called waste paper.
- old books, magazines and used notebook pages, flyers, envelopes, stationery, postcards, tissue paper, and the like can be used.
- the concentration of the pulp in the liquid medium is preferably 2% W / V or more.
- the pulp concentration is less than 2% W / V, the production amount of cellulase, particularly ⁇ -glucanase may not increase so much.
- the upper limit is an amount that allows the liquid medium to be stirred and mixed.
- the paper is preferably cut and used with a shredder.
- the upper limit of the initial concentration of pulp in the liquid medium can be 20, 15 or 10% W / V depending on the performance of the agitator.
- the preferred range of initial pulp concentration is 2-7% W / V, preferably 3-5% 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, sodium glutamate).
- 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 35 to 660 mM in terms of moles of ammonium. Preferably, it is 50 to 580 mM. When the concentration is less than 35 mM, the production amount of cellulase, particularly ⁇ -glucanase may not increase so much. Further, when the initial concentration exceeds 660 mM, the productivity of the enzyme decreases.
- the concentration of ammonia nitrogen or amino nitrogen in the liquid medium is preferably increased or decreased according to the pulp concentration in the liquid medium. For example, when the pulp concentration is 3% W / V, Considering cost etc., 50 mM is preferable.
- Trichoderma filamentous fungi Process for producing ⁇ -glucanase and xylanase Trichoderma filamentous fungi are known to produce cellulases necessary for saccharification of cellulose.
- the microorganism belonging to the genus Trichoderma used in the present invention is not particularly limited as long as it produces cellulase.
- a preferred microorganism belonging to the genus Trichoderma is Trichoderma reesei or Trichoderma viride. Particularly preferred is Trichoderma reesei.
- the bacteriological properties of the fungi Trichoderma reesei and Trichoderma viride include, for example, EG Simmons, Abstract Second International Mycological Congress (EG (Simmons, Abst. 2nd International Mycological Congress) Miami, Florida, USA (March 1977, p. 618).
- 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.
- the additional form may be continuous or batchwise, and the additional time and amount may be adjusted so that stirring and mixing are possible even after the pulp is added.
- ammonia nitrogen or amino nitrogen may be added as needed.
- the Trichoderma filamentous fungus culture solution or culture supernatant contains a high concentration of the target cellulase, that is, ⁇ -glucanase and xylanase.
- the ⁇ -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 even more preferably 70 U / ml or more.
- 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.
- the said hemicellulase activity can be quantified by making the reducing sugar produced
- 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 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.
- 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 ⁇ mol of xylose per minute under the reaction conditions of 40 ° C. and 10 minutes.
- ⁇ -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 ⁇ -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.
- the cellulose resource is suspended in an aqueous medium as a substrate, and the above culture solution or culture medium is used.
- a saccharification reaction is performed by adding a clear liquid and heating the mixture while stirring or shaking.
- 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.
- the pH and temperature of the reaction solution may be within the range where the enzyme is not inactivated.
- 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.
- 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.
- Example 1 Trichoderma reesei QM9414 (NBRC 31329) was cultured on potato dextrose agar at 28 ° C. for 7 days to sufficiently form spores.
- the crystalline cellulose which is the carbon source of Mandel's medium is replaced with 3% (3 g / 100 ml) of copy paper, and the ammonium nitrogen concentration of ammonium sulfate which is the nitrogen source is 15 mM, 35 mM, 50 mM, 65 mM, 80 mM, 100 mM or
- a 500 ml baffled Erlenmeyer flask was prepared with 100 ml of liquid medium added to 115 mM and adjusted to pH 4.8 with phosphoric acid and sodium hydroxide.
- the enzyme activity of the culture solution obtained above was measured.
- the ⁇ -glucanase activity was determined by measuring the absorbance of a stained fragment produced by enzymatic degradation using a ⁇ -glucan labeled with a substrate using a ⁇ -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% 0.6% zinc acetate and 80% methyl cellosolve (pH 5)] was added and left for 5 minutes to stop the reaction.
- ⁇ -glucanase activity was expressed as the amount of enzyme that produces reducing sugar corresponding to 1 ⁇ mol of glucose per minute under the reaction conditions of 40 ° C. and 10 minutes.
- the xylanase activity was quantified by increasing the absorbance at 540 nm by reacting the reducing sugar produced by enzymatic hydrolysis with xylan derived from oat spelts as a substrate. 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 the solution was accurately obtained at 40 ° C.
- DNS reagent (0.75% dinitrosalicylic acid, 1.2% sodium hydroxide, 22.5% sodium potassium tartrate tetrahydrate, 0.3% lactose monohydrate 4 ml) was added and mixed well to stop the reaction.
- the reaction stop solution was accurately heated in a boiling water bath for 15 minutes.
- 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 ⁇ mol of xylose per minute under the reaction conditions of 40 ° C. and 10 minutes. The results are shown in FIG.
- Example 2 A liquid medium was prepared in the same manner as in Example 1 except that crystalline cellulose, which is the carbon source of the Mandel medium, was replaced with cardboard 3% (3 g / 100 ml).
- 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 ⁇ -glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.
- Example 3 A liquid medium was prepared in the same manner as in Example 1 by replacing crystalline cellulose, which is the carbon source of the Mandel medium, with 3% (3 g / 100 ml) of newspaper.
- 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 ⁇ -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, which is the carbon source of Mandel's medium, is replaced with 3% (3 g / 100 ml) of copy paper, and ammonium sulfate, which is the nitrogen source, is replaced with ammonium chloride so that the molar concentrations of ammonia nitrogen are 20 mM, 40 mM, 50 mM, and 60 mM, respectively.
- a liquid medium was prepared by adding 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 ⁇ -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 Mandel's medium is replaced with 3% (3 g / 100 ml) of copy paper, and ammonium sulfate that is the nitrogen source is replaced with diammonium phosphate, so that the molar concentration of ammonia nitrogen is 15 mM, 35 mM, and 50 mM, respectively.
- a liquid medium was prepared in the same manner as in Example 1 by adding 65 mM, 80 mM, 100 mM, or 115 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 ⁇ -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, which is the carbon source of Mandel's medium, is replaced with 3% (3 g / 100 ml) of copy paper, and ammonium sulfate, which is the nitrogen source, is replaced with ammonium nitrate so that the molar concentration of ammonia nitrogen is 12 mM, 24 mM, 36 mM, 48 mM, A liquid medium was prepared in the same manner as in Example 1 by adding 60 mM, 72 mM, or 84 mM. Trichoderma reesei QM9414 (NBRC 31329) is cultured on potato dextrose agar medium at 28 ° C. for 7 days to fully form spores.
- NBRC 31329 Trichoderma reesei QM9414
- 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 ⁇ -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 that is the carbon source of Mandel's medium is replaced with 3% (3 g / 100 ml) of copy paper, and the ammonium sulfate that is the nitrogen source is replaced with aqueous ammonia, so that the molar concentrations are 15 mM, 30 mM, 45 mM, 65 mM, 75 mM, and 90 mM, respectively.
- a liquid medium was prepared in the same manner as in Example 1 by adding to 105 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 ⁇ -glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.
- Example 8 The crystalline cellulose which is the carbon source of Mandel's medium is replaced with 3% (3 g / 100 ml) of copy paper, and ammonium sulfate which is the nitrogen source is replaced with urea, so that the molar concentration of amino nitrogen is 17 mM, 33 mM, 50 mM, 67 mM, A liquid medium was prepared in the same manner as in Example 1 by adding 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.
- NBRC 31329 Trichoderma reesei QM9414
- 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 ⁇ -glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.
- Example 9 Replace the crystalline cellulose, which is the carbon source of Mandel's medium, with copy paper and add it to a concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and it is also a nitrogen source
- a liquid medium was prepared in the same manner as in Example 1 by adding ammonium sulfate so that the molar concentration of ammonia nitrogen was 80 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 ⁇ -glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.
- Example 10 Replace the crystalline cellulose, which is the carbon source of Mandel's medium, with copy paper and add it to a concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 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 ⁇ -glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.
- Example 11 Replace the crystalline cellulose, which is the carbon source of Mandel's medium, with copy paper and add it to a concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 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 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 ⁇ -glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.
- Example 12 Replace the crystalline cellulose, which is the carbon source of Mandel's medium, with copy paper and add it to a concentration of 1%, 2%, 3%, 4%, 5%, 6%, 7%, and 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 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 ⁇ -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 ⁇ -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 ⁇ -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 Mandel's medium, is added in place of copy paper 1% (3 g / 100 ml), and the molar concentration of ammonium nitrogen as the nitrogen source is 15 mM, 35 mM, 50 mM, 65 mM, 80 mM, respectively.
- a liquid medium was prepared.
- 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 ⁇ -glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.
- Reference example 4 Crystalline cellulose, which is the carbon source of Mandel's medium, is added by replacing with 3% (3 g / 100 ml) of copy paper, and ammonium sulfate, which is the nitrogen source, is replaced by potassium nitrate, so that the molarity is 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, A liquid medium was prepared in the same manner as in Example 1 by adding 60 mM or 70 mM. Trichoderma reesei QM9414 (NBRC 31329) is cultured on potato dextrose agar medium at 28 ° C. for 7 days to fully form spores.
- NBRC 31329 Trichoderma reesei QM9414
- 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 ⁇ -glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.
- Example 13 Trichoderma reesei QM9414 (NBRC 31329) was cultured on potato dextrose agar at 28 ° C. for 7 days to sufficiently form spores.
- the 1 platinum loop was inoculated into a 500 ml baffled Erlenmeyer flask containing 100 ml of Mandel medium, and cultured with shaking at 28 ° C., 180 rpm for 4 days.
- a 500 ml baffled Erlenmeyer flask containing 150 ml of Mandel medium was inoculated and cultured with shaking at 28 ° C., 180 rpm for 2 days to obtain a culture solution.
- crystalline cellulose which is the carbon source of Mandel's medium
- 3% (30 g / l), 6% (60 g / l) of copy paper or 3% of crystalline cellulose (Avicel PH101) is added, and ammonium sulfate, which is the nitrogen source, is added.
- Ammonia nitrogen was added to a molar concentration of 200 mM, and 3 L of medium using 6 g / l of Adecanol LG-126 (manufactured by ADEKA Co., Ltd.) instead of Tween 80 and 5 L fermenter (manufactured by Marubishi) And cultured at 28 ° C.
- Aeration was 1 VVM, agitation was 450 rpm, and the pH was adjusted to be constant at pH 4.8 during the culture period with 2N sodium hydroxide and 5-fold diluted phosphoric acid.
- the culture broth was centrifuged, and ⁇ -glucanase activity and xylanase activity of the supernatant were measured. The result is shown in FIG.
- the copy paper was cut into 4 mm ⁇ 30 mm using a shredder (Primo 1400 manufactured by Meiko Trading Company). Further, when 6% (60 g / l) of copy paper was added all at once, stirring was insufficient, so copy paper and ammonium sulfate were added in half on the first and third days of culture.
- a shredder Principal 1400 manufactured by Meiko Trading Company.
- Example 14 In the same manner as in Example 13, 6% (60 g / l) of copy paper was added to a 5 L fermentor, and the ammonium sulfate was added so that the molar concentration of ammonia nitrogen was 44 mM, 100 mM, 134 mM, or 224 mM. Investigated production. The result is shown in FIG.
- Example 15 In the same manner as in Example 13, 6% (60 g / l) of copy paper and 45 mM of ammonium nitrogen molar concentration of ammonium sulfate were added to a 5 L fermentor, and the pH adjustment chemical solution during the culture period was added with 2N sodium hydroxide. The enzyme production was compared with the case of using 18% and 18% aqueous ammonia. At this time, the input amount of ammonia water during the culture period was 123 mM. The result is shown in FIG.
- Example 16 The crystalline cellulose which is the carbon source of Mandel's medium is replaced with 3% (3 g / 100 ml) of copy paper, and the ammonium nitrogen which is the nitrogen source has a molar concentration of ammonia nitrogen of 330 mM, 420 mM, 500 mM, 580 mM, 660 mM, 720 mM or A liquid medium was prepared in the same manner as in Example 1 by adding to 800 mM. Trichoderma reesei QM9414 (NBRC 31329) is cultured on potato dextrose agar medium at 28 ° C. for 7 days to fully form spores.
- NBRC 31329 Trichoderma reesei QM9414
- 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 ⁇ -glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.
- Example 17 The crystalline cellulose that is the carbon source of Mandel's medium is replaced with 3% (3 g / 100 ml) of copy paper, and ammonium sulfate that is the nitrogen source is replaced with sodium glutamate, so that the molar concentration of amino nitrogen is 17 mM, 33 mM, 50 mM, and 67 mM, respectively. , 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.
- NBRC 31329 Trichoderma reesei QM9414
- 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 ⁇ -glucanase activity and xylanase activity were measured in the same manner as in Example 1. The results are shown in FIG.
- Example 18 A saccharification test of cellulose resources was carried out using the supernatants of the 6% copy paper medium and the 3% crystalline cellulose medium obtained in Example 13, respectively.
- Cellulose resources used for saccharification were cellulose powder (trade name KC Flock W-50 manufactured by Nippon Paper Chemicals), bagasse, rice straw and beer lees. Bagasse, rice straw and beer lees are finely pulverized, suspended in 0.3N NaOH, treated at 120 ° C. for 15 minutes, thoroughly washed with water, dried, and delignified. It was subjected to saccharification. The cellulose powder was subjected to saccharification as it was.
- ⁇ -glucanase and xylanase were obtained by culturing Trichoderma reesei using a liquid medium containing untreated paper as a carbon source and ammonia / ammonium salt as a nitrogen source. At the same time, it was shown to be highly productive. In addition, by increasing the concentration of papers in the medium from the commonly used carbon source, or by adjusting the ammonia / ammonium salt concentration in the medium to a specific range, the amount of cellulase produced may be significantly increased. Indicated. Furthermore, it was shown that various cellulose resources can be decomposed and saccharified using the obtained culture supernatant.
- ⁇ -glucanase and xylanase which are extremely useful for saccharification of cellulose resources, especially 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.
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Abstract
Description
本発明の液体培地はトリコデルマ属に属する微生物が生育する栄養を含む材料である。かかる液体培地は、以下の培地組成を水100mlに溶解及び懸濁した液体培地(一般に、マンデル培地と呼ばれる)を基に調整され、炭素源としてパルプ、及び窒素源としてアンモニア態窒素又はアミノ態窒素を含むものである。好ましい培地組成の一例を以下に示す。
トリコデルマ属糸状菌はセルロースの糖化に必要なセルラーゼの生産菌として知られている。本発明に使用するトリコデルマ属に属する微生物はセルラーゼを生産するものであれば特に限定されない。好ましいトリコデルマ属に属する微生物はトリコデルマ・リーセイ又はトリコデルマ・ビリデである。特に好ましくは、トリコデルマ・リーセイである。
本発明の方法により得られたβ-グルカナーゼ及びキシラナーゼは、セルロース資源を分解または糖化するのに有用である。ここでいうセルロース資源は、合成セルロースもしくは天然セルロース資源のどちらでも良い。合成セルロースとは、セルロース粉末として、流通しているものを表す。天然セルロース資源とは、バガス、稲わら、麦わら、ビール粕、木材などが挙げられる。本発明は、β-グルカナーゼおよびキシラナーゼを同時に高生産できるため、特に、バガス、稲わら、麦わら、ビール粕などの天然セルロース資源の糖化に優れている。
トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させた。マンデル培地の炭素源である結晶セルロースをコピー用紙3%(3g/100ml)に置き換えて、また窒素源である硫酸アンモニウムのアンモニア態窒素のモル濃度がそれぞれ15mM、35mM、50mM、65mM、80mM、100mMまたは115mMになるように添加して燐酸及び水酸化ナトリウムでpH4.8に調整した100mlの液体培地を500ml容バッフル付三角フラスコに用意した。培養したトリコデルマ・リーセイの1白金耳をこの液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、上清液のβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。なお、コピー用紙はアルカリ処理や熱処理等の前処理をせず、シュレッダー(カール社デスクパーサーDS-4100)で2mmX7mmに裁断したのみで使用した。
前記で得られた培養液について酵素活性を測定した。
β-グルカナーゼ活性は、メガザイム社製のβ‐グルカナーゼ測定キットを用い、色素標識したβ-グルカンを基質とした酵素分解によって生じた染色断片を吸光度測定した。具体的には、アゾ大麦グルカン基質溶液0.1mlに培養液0.1mlを加えて、40℃にて正確に10分間酵素反応を行なわせた後、停止液〔4%酢酸ナトリウム、0.4%酢酸亜鉛、80%メチルセルソルブを含む(pH5)〕0.6mlを加えて5分放置し、反応を停止した。続いて遠心分離した後、上澄液を590nmの吸光度測定した。1単位のβ-グルカナーゼ活性は、40℃、10分間の反応条件下で、1分間に1μmolのグルコースに相当する還元糖を生成する酵素量として表した。
マンデル培地の炭素源である結晶セルロースをダンボール3%(3g/100ml)に置き換えて、実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図2に示す。
マンデル培地の炭素源である結晶セルロースを新聞紙3%(3g/100ml)に置き換えて、実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図3に示す。
マンデル培地の炭素源である結晶セルロースをコピー紙3%(3g/100ml)に置き換えて、また窒素源である硫酸アンモニウムを塩化アンモニウムに置き換えてアンモニア態窒素のモル濃度がそれぞれ20mM、40mM、50mM、60mM、80mM、100mMまたは120mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図4に示す。
マンデル培地の炭素源である結晶セルロースをコピー紙3%(3g/100ml)に置き換えて、また窒素源である硫酸アンモニウムを燐酸二アンモニウムに置き換えてアンモニア態窒素のモル濃度がそれぞれ15mM、35mM、50mM、65mM、80mM、100mMまたは115mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図5に示す。
マンデル培地の炭素源である結晶セルロースをコピー紙3%(3g/100ml)に置き換えて、また窒素源である硫酸アンモニウムを硝酸アンモニウムに置き換えてアンモニア態窒素のモル濃度がそれぞれ12mM、24mM、36mM、48mM、60mM、72mMまたは84mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図6に示す。
マンデル培地の炭素源である結晶セルロースをコピー紙3%(3g/100ml)に置き換えて、また窒素源である硫酸アンモニウムをアンモニア水に置き換えてモル濃度がそれぞれ15mM、30mM、45mM、65mM、75mM、90mMまたは105mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図7に示す。
マンデル培地の炭素源である結晶セルロースをコピー紙3%(3g/100ml)に置き換えて、また窒素源である硫酸アンモニウムを尿素に置き換えてアミノ態窒素のモル濃度がそれぞれ17mM、33mM、50mM、67mM、83mMまたは100mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図8に示す。
マンデル培地の炭素源である結晶セルロースをコピー紙に置き換えて濃度が1%、2%、3%、4%、5%、6%、7%になるように添加して、また窒素源である硫酸アンモニウムのアンモニア態窒素のモル濃度が80mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図9に示す。
マンデル培地の炭素源である結晶セルロースをコピー紙に置き換えて濃度が1%、2%、3%、4%、5%、6%、7%になるように添加して、また窒素源である硫酸アンモニウムのアンモニア態窒素のモル濃度が160mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図10に示す。
マンデル培地の炭素源である結晶セルロースをコピー紙に置き換えて濃度が1%、2%、3%、4%、5%、6%、7%になるように添加して、また窒素源である硫酸アンモニウムのアンモニア態窒素のモル濃度が320mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図11に示す。
マンデル培地の炭素源である結晶セルロースをコピー紙に置き換えて濃度が1%、2%、3%、4%、5%、6%、7%になるように添加して、また窒素源である硫酸アンモニウムのアンモニア態窒素のモル濃度が480mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図12に示す。
マンデル培地の炭素源である結晶セルロースの濃度を1%とし、窒素源である硫酸アンモニウムのアンモニア態窒素のモル濃度がそれぞれ15mM、35mM、50mM、65mM、80mM、100mMまたは115mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図13に示す。
マンデル培地の炭素源である結晶セルロース濃度が1%、1.5%、2%、2.5%、3%、3.5%または4%になるように添加して、また窒素源である硫酸アンモニウムのアンモニア態窒素のモル濃度が160mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図14に示す。
マンデル培地の炭素源である結晶セルロースをコピー紙1%(3g/100ml)に置き換えて添加して、また窒素源である硫酸アンモニウムのアンモニア態窒素のモル濃度がそれぞれ15mM、35mM、50mM、65mM、80mM、100mMまたは115mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図15に示す。
マンデル培地の炭素源である結晶セルロースをコピー紙3%(3g/100ml)に置き換えて添加して、また窒素源である硫酸アンモニウムを硝酸カリウムに置き換えてモル濃度が10mM、20mM、30mM、40mM、50mM、60mMまたは70mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図16に示す。
トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させた。その1白金耳を、マンデル培地100mlを含む500ml容バッフル付三角フラスコに接種して、28℃、180rpm、4日間振とう培養した。マンデル培地150mlを含む500ml容バッフル付三角フラスコに接種して、28℃、180rpm、2日間振とう培養して培養液を得た。マンデル培地の炭素源である結晶セルロースに置き換えて、コピー紙3%(30g/l)、6%(60g/l)又は結晶セルロース(アビセルPH101)3%を添加し、また窒素源である硫酸アンモニウムのアンモニア態窒素のモル濃度が200mMになるように添加し、ツイーン80のかわりにアデカノールLG-126(株式会社ADEKA製)6g/lを用いた培地3L及び本培養液を5L発酵槽(マルビシ製)に添加して、28℃で培養した。通気は1VVM、撹拌は450rpmで、またpHは2N水酸化ナトリウムおよび5倍希釈の燐酸によって培養期間中pH4.8に一定になるよう調整した。7日目に培養液を遠心分離し、その上清液のβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。その結果を図17に示す。
実施例13と同様の方法で5L発酵槽に、コピー紙6%(60g/l)を添加し、硫酸アンモニウムのアンモニア態窒素のモル濃度が44mM、100mM、134mMまたは224mMになるように添加し、酵素生産を調べた。その結果を図18に示す。
実施例13と同様の方法で5L発酵槽に、コピー紙6%(60g/l)と硫酸アンモニウムのアンモニア態窒素のモル濃度が45mMを添加し、培養期間中のpH調整用薬液を2N水酸化ナトリウムを使用した場合と、18%アンモニア水を使用した場合の酵素生産を比較した。このときのアンモニア水の培養期間中の投入量は123mMであった。その結果を図19に示す。
マンデル培地の炭素源である結晶セルロースをコピー紙3%(3g/100ml)に置き換えて、また窒素源である硫酸アンモニウムのアンモニア態窒素のモル濃度がそれぞれ330mM、420mM、500mM、580mM、660mM、720mMまたは800mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図20に示す。
マンデル培地の炭素源である結晶セルロースをコピー紙3%(3g/100ml)に置き換えて、また窒素源である硫酸アンモニウムをグルタミン酸ナトリウムに置き換えてアミノ態窒素のモル濃度がそれぞれ17mM、33mM、50mM、67mM、83mMまたは100mMになるように添加して実施例1と同様に液体培地を用意した。トリコデルマ・リーセイQM9414(NBRC 31329)をポテトデキストロース寒天培地上で28℃、7日間培養して胞子を充分形成させ、この1白金耳を液体培地に接種して、28℃、180rpm、7日間振とう培養した。7日目に培養液を遠心分離し、実施例1と同様にしてβ-グルカナーゼ活性およびキシラナーゼ活性を測定した。結果を図21に示す。
実施例13で得られたコピー紙6%の培地及び結晶セルロース3%の培地のそれぞれの上清液を用いて、セルロース資源の糖化試験を行った。糖化に供するセルロース資源は、セルロース粉末(日本製紙ケミカル製 商品名KCフロックW-50)、バガス、稲わらおよびビール粕を用いた。また、バガス、稲わらおよびビール粕は、それぞれ微粉砕し、0.3NのNaOHに懸濁して、120℃、15分間処理し、水で充分に洗浄後、乾燥し、脱リグニン処理を行い、糖化に供した。セルロース粉末は、そのまま糖化に供した。セルロース資源:0.8g、培養上清液:9ml、1M酢酸バッファー(pH4.8):0.2mlからなる液(セルロース資源8%液)を50℃、pH4.8、48時間、振とうさせて糖化し、生成したグルコースをグルコースCII-テストワコー(和光純薬工業)で測定した。結果を図22~図25に示す。
Claims (13)
- (a)炭素源として加熱処理もアルカリ処理も行わない紙類由来のパルプ、及び(b)窒素源としてアンモニア態窒素またはアミノ態窒素を含む液体培地を用いて、トリコデルマ属に属する微生物を培養する工程を包含するβ-グルカナーゼ及びキシラナーゼの製造方法。
- 前記パルプの前記液体培地中における初期濃度が2%W/V以上である請求項1に記載のβ-グルカナーゼ及びキラナーゼの製造方法。
- 前記パルプの前記液体培地中における初期濃度が2~7%W/Vである請求項1又は2に記載のβ-グルカナーゼ及びキシラナーゼの製造方法。
- 前記アンモニア態窒素またはアミノ態窒素の前記液体培地中における初期濃度が35~660mM以上である請求項1~3のいずれかに記載のβ-グルカナーゼ及びキシラナーゼの製造方法。
- 前記アンモニア態窒素またはアミノ態窒素の前記液体培地中における初期濃度が50~580mMである請求項1~4のいずれかに記載のβ-グルカナーゼ及びキシラナーゼの製造方法。
- 前記紙類が上質紙、更紙、コピー用紙、新聞紙及びダンボール紙からなる群から選択される少なくとも一種である請求項1~5のいずれかに記載のβ-グルカナーゼ及びキシラナーゼの製造方法。
- 前記トリコデルマ属に属する微生物が、トリコデルマ・リーセイである請求項1~6のいずれかに記載のβ-グルカナーゼ及びキシラナーゼの製造方法。
- 培養の過程において前記液体培地に対してパルプを追加する請求項1~7に記載のβ-グルカナーゼ及びキシラナーゼの製造方法。
- (a)炭素源として加熱処理もアルカリ処理も行わない紙類由来のパルプ、及び(b)窒素源としてアンモニア態窒素またはアミノ態窒素を含む液体培地であって、トリコデルマ属に属する微生物を培養するために用いられる液体培地。
- 前記パルプを2%W/V以上含有する請求項9に記載の液体培地。
- アンモニア態窒素またはアミノ態窒素を35~660mM含有する請求項9又は10に記載の液体培地。
- 請求項1~8のいずれか1項に記載の方法により製造されたβ-グルカナーゼ及びキシラナーゼ。
- 請求項12記載のβ-グルカナーゼ及びキシラナーゼを用いることを特徴とするセルロース資源の分解または糖化方法。
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