WO2016171215A1 - Method for producing fumaric acid - Google Patents

Method for producing fumaric acid Download PDF

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WO2016171215A1
WO2016171215A1 PCT/JP2016/062641 JP2016062641W WO2016171215A1 WO 2016171215 A1 WO2016171215 A1 WO 2016171215A1 JP 2016062641 W JP2016062641 W JP 2016062641W WO 2016171215 A1 WO2016171215 A1 WO 2016171215A1
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rhizopus
medium
fumaric acid
culture
sorbitol
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PCT/JP2016/062641
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French (fr)
Japanese (ja)
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史員 高橋
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花王株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/44—Polycarboxylic acids
    • C12P7/46—Dicarboxylic acids having four or less carbon atoms, e.g. fumaric acid, maleic acid

Definitions

  • the present invention relates to a method for producing fumaric acid.
  • Fumaric acid is often used as an acidulant and antibacterial agent, and is also used in food production as an intermediate substance such as malic acid and aspartic acid. It is also used as a raw material for synthetic resins and biodegradable polymers. It is a highly valuable substance.
  • fumaric acid has been produced using chemical synthesis methods and fermenting bacteria such as Rhizopus. Previously, chemical synthesis methods were the mainstream because of their lower cost, but in recent years, microbiological methods have attracted attention again from the viewpoints of soaring raw materials, safety during food use, environmental impact, etc. .
  • Fumaric acid is known as an intermediate substance in the TCA cycle, but in Rhizopus sp., It is considered that it is produced in a reductive TCA pathway starting from pyruvic acid generated mainly from glucose. On the other hand, in Rhizopus sp., There is a pathway for producing ethanol from pyruvic acid. Therefore, the production of fumaric acid by Rhizopus sp has a problem that ethanol is mixed as a by-product.
  • Non-patent Document 1 Mutant Rhizopus spp. With high fumaric acid production and low ethanol production have been reported (Non-patent Document 1). However, since the genetic background of Rhizopus spp. Has not yet been fully studied, it is not easy to develop a high fumarate-producing Rhizopus spp.
  • Patent Document 1 discloses a method for producing an organic acid in which Rhizopus is cultured in a medium to which a fatty acid ester or triglyceride is added.
  • Patent Document 2 discloses that Aspergillus genus bacteria were cultured in a medium to which various sugars other than glucose were added, and malic acid productivity was examined. However, the malic acid productivity in the case of using sugars other than glucose was lower than that of glucose.
  • Patent Document 3 discloses that succinic acid was produced by various bacteria such as Actinobacillus succinogenes from crude sorbitol produced from biomass.
  • Patent Document 4 discloses that succinic acid was produced from sorbitol in recombinant Actinobacillus succinogenes.
  • Non-Patent Document 2 discloses that succinic acid productivity was improved by culturing recombinant Escherichia coli in a sorbitol-added medium as compared to a glucose-added medium, but at the same time, ethanol production was also increased.
  • Patent Literature 1 US Pat. No. 4,564,594 (Patent Literature 2) British Patent No. 884029 (Patent Literature 3) US Patent Publication No. 2014/0093926 (Patent Literature 4) US Patent Publication No. 2014/0093925 (Non-Patent Document 1) Korean J Chem Eng, 2010, 27 (1): 183-186 (Non-patent document 2) J Ind Microbiol Biotechnol, 2005, 32 (3): 87-93
  • the present invention provides a method for producing fumaric acid, comprising culturing Rhizopus sp. In a medium containing sorbitol.
  • the present invention relates to an efficient microbiological production method of fumaric acid.
  • the present inventors have improved the fumaric acid productivity of the fungus by culturing Rhizopus sp. In a medium containing sorbitol as a carbon source, and It has been found that the production of ethanol as a by-product is suppressed.
  • the yield of fumaric acid can be increased and the production of ethanol as a byproduct can be suppressed as compared with the conventional method of producing fumaric acid by Rhizopus sp. Therefore, according to the present invention, fumaric acid can be produced more efficiently and at low cost.
  • Rhizopus is cultured in a medium containing sorbitol. After completion of the culture, the produced fumaric acid is recovered from the medium.
  • Rhizopus sp. (1.1. Bacterial species)
  • the type of Rhizopus genus used in the method for producing fumaric acid of the present invention is not particularly limited, and examples thereof include Rhizopus delemar, Rhizopus oryzae, Rhizopus ariziz, Rhizopus chinhis, . These Rhizopus species may be used alone or in combination of two or more.
  • the Rhizopus bacterium is preferably used in the form of a pellet.
  • the Rhizopus bacterium may be a Rhizopus bacterium immobilized on a carrier, or the pellet and the bacterium immobilized on the carrier may be used in combination.
  • a “pellet” of Rhizopus sp. Means a hypha mass having a size of several hundred ⁇ m to several mm spontaneously formed by hyphae by liquid culture, and “Rhizopus sp. "Means Rhizopus spp. Held or embedded in a carrier.
  • the Rhizopus genus bacteria immobilized on the Rhizopus bacterium pellets and the carrier may be those obtained commercially or those prepared from spores or hyphae.
  • Rhizopus pellets can be prepared by a known method. For example, a spore suspension of Rhizopus spp. Is prepared (Step A), and it is cultured in a culture solution to germinate spores to prepare a mycelium (Step B), preferably obtained in Step B. The obtained mycelium is grown (step C) to form a pellet.
  • the prepared pellet can be separated and recovered from the culture solution by filtration, centrifugation, etc., and used for fumaric acid production. A preferred embodiment of steps A to C is described in detail below.
  • spore suspension Preparation of spore suspension> The spores of Rhizopus sp., For example, an inorganic agar medium (composition example: 2% glucose, 0.1% ammonium sulfate, 0.06% potassium dihydrogen phosphate, 0.025% magnesium sulfate heptahydrate, 0. Inoculate 009% zinc sulfate heptahydrate, 1.5% agar (both concentrations are% (w / v)), PDA medium, etc., at 10-40 ° C, preferably 27-30 ° C
  • the spore suspension can be prepared by performing stationary culture for 7 to 10 days and then suspending in physiological saline or the like.
  • the spore suspension may or may not contain mycelium.
  • ⁇ Step B Preparation of mycelium>
  • the spore suspension obtained above is inoculated into a culture solution and cultured, and the spores are germinated to obtain mycelium.
  • the culture may be performed according to a normal procedure.
  • the number of spores of the filamentous fungus inoculated into the culture solution is 1 ⁇ 10 2 to 1 ⁇ 10 8 spore / mL-culture solution, preferably 1 ⁇ 10 2 to 5 ⁇ 10 4 spore / mL-culture solution, More preferred is 5 ⁇ 10 2 to 1 ⁇ 10 4 spores / mL-culture solution, and further preferred is 1 ⁇ 10 3 to 1 ⁇ 10 4 spores / mL-culture solution.
  • the culture solution may be a commercially available medium, for example, potato dextrose medium (hereinafter also referred to as PDB medium, for example, Becton Dickinson and Company), Luria-Bertani medium (hereinafter also referred to as LB medium, for example, Nippon Pharmaceutical).
  • PDB medium potato dextrose medium
  • LB medium Luria-Bertani medium
  • SB medium for example, manufactured by OXOID
  • SB medium for example, manufactured by OXOID
  • the culture solution may be a monosaccharide such as glucose or xylose as a carbon source, an oligosaccharide such as sucrose, lactose or maltose, or a polysaccharide such as starch; glycerin, citric acid or the like.
  • Biological components ammonium sulfate, urea, etc. as a nitrogen source; various salts such as sodium, potassium, magnesium, zinc, iron, phosphoric acid, etc. can be added as appropriate.
  • the preferred concentration of monosaccharide, oligosaccharide, polysaccharide and glycerin is 0.1-30% (w / v)
  • the preferred concentration of citric acid is 0.01-10% (w / v)
  • the preferred concentration of nitrogen source is 0 .01 to 1% (w / v)
  • the preferred concentration of the inorganic substance is 0.0001 to 0.5% (w / v).
  • the culture broth is inoculated with a spore suspension, and is preferably stirred at 80 to 250 rpm, more preferably 100 to 170 rpm, under a culture temperature control of 25 to 42.5 ° C., preferably for 24 to 120 hours.
  • the culture is preferably performed for 48 to 72 hours.
  • the amount of the culture solution used for the culture may be appropriately adjusted according to the culture vessel. For example, it may be about 50 to 100 mL for a 200 mL baffled flask and about 100 to 300 mL for a 500 mL baffled flask. That's fine.
  • the inoculated spores germinate and grow into mycelium.
  • Step C is a step of further culturing and growing the mycelium obtained in Step B. Although this step is not essential, it is preferable to perform this step from the viewpoint of improving the fumaric acid productivity.
  • the growth medium used in Step C is not particularly limited, and may be any inorganic culture medium containing glucose that is usually used.
  • 7.5 to 30% glucose, 0.05 to 2% ammonium sulfate, 0 0.03-0.6% potassium dihydrogen phosphate, 0.01-0.1% magnesium sulfate heptahydrate, 0.005-0.05% zinc sulfate heptahydrate, and 3.75- Examples include a culture solution containing 20% calcium carbonate (both concentrations are% (w / v)).
  • the amount of the culture solution may be appropriately adjusted according to the culture vessel. For example, in the case of a 500 mL Erlenmeyer flask, it may be 50 to 300 mL, preferably 100 to 200 mL.
  • the cells cultured in step B are inoculated in a wet weight of 1 to 6 g-cells / 100 mL-culture solution, preferably 3 to 4 g-cells / 100 mL-culture solution, and 100 to 100-
  • the culture is performed for 12 to 120 hours, preferably 24 to 72 hours under a culture temperature control of 25 to 42.5 ° C. while stirring at 300 rpm, preferably 170 to 230 rpm.
  • Rhizopus bacterium immobilized on the carrier can be prepared by a known method.
  • the spores of Rhizopus sp. Are inoculated into a liquid medium containing a carrier and cultured, and the carrier in which the mycelium germinated from the spore is taken is recovered (see JP 2013-240321 A).
  • Examples of the material of the carrier for immobilizing Rhizopus sp. include urethane polymers, olefin polymers, diene polymers, condensation polymers, silicone polymers, fluorine polymers, and the structure thereof.
  • the shape of the carrier may be any of a flat plate shape, a multilayer plate shape, a corrugated plate shape, a tetrahedron shape, a spherical shape, a string shape, a net shape, a columnar shape, a lattice shape, a cylindrical shape, and the size is the length of the longest side.
  • the thickness is preferably 0.1 mm to 10 mm, more preferably 0.3 to 5 mm, and even more preferably 0.5 to 2 mm.
  • the Rhizopus bacterium-immobilized carrier can be separated and recovered from the culture solution by filtration, centrifugation, or the like, and used for fumaric acid production.
  • the medium As a medium used for the fumaric acid production method of the present invention, a medium usually used for organic acid production by Rhizopus sp. May be used except that the carbon source contains sorbitol.
  • the medium is a liquid medium, and may be any of a synthetic medium, a natural medium, and a semi-synthetic medium obtained by adding a natural component to a synthetic medium.
  • the medium generally contains a carbon source, a nitrogen source, an inorganic salt, and the like, but each component composition can be appropriately selected except for the above-described carbon source.
  • the preferable medium composition is described in detail below.
  • concentration of each component in the culture medium described below represents the density
  • the above medium contains sorbitol.
  • the amount of sorbitol contained in the medium is preferably 10% by mass or more, more preferably 10 to 100% by mass, and still more preferably 50 to 100% by mass in the total amount of the carbon source contained in the medium.
  • the carbon source of the medium is a saccharide such as sugar or sugar alcohol, and is preferably 10% by mass or more, more preferably 10 to 100% by mass, and still more preferably 50 to 100% in the total amount of the saccharide.
  • Mass% sorbitol is included.
  • the medium may contain a carbon source other than the saccharide, for example, a fatty acid ester or a triglyceride.
  • the amount of sorbitol in the medium is preferably 10% by mass or more in the saccharide. More preferably, it is 10 to 100% by mass, and still more preferably 50 to 100% by mass.
  • glucose / (sorbitol + glucose) is preferably in the range of 0 to 0.5, more preferably 0 to 0.3, and even more preferably 0 to 0.1. .
  • the saccharide that is the carbon source of the medium may contain a saccharide other than sorbitol.
  • saccharides other than sorbitol include glucose, maltose, starch hydrolysate, fructose, xylose, xylitol, sucrose and the like, among which glucose, fructose and xylitol are preferred. These saccharides can be used alone or in combination of two or more.
  • the concentration of the carbon source (preferably saccharide containing sorbitol) in the medium is preferably 1% (w / v) or more, more preferably 5% (w / v) or more, and preferably 40% ( w / v) or less, more preferably 30% (w / v) or less.
  • the concentration of the carbon source (including sorbitol) in the medium is preferably 1 to 40% (w / v), more preferably 5 to 30% (w / v).
  • Examples of the nitrogen source in the medium include nitrogen-containing compounds such as urea, ammonium nitrate, potassium nitrate, and sodium nitrate.
  • the concentration of the nitrogen source in the medium is preferably 0.001 to 0.5% (w / v), more preferably 0.05 to 0.2% (w / v).
  • the above medium can contain sulfate, magnesium salt, zinc salt and the like.
  • sulfates include magnesium sulfate, zinc sulfate, potassium sulfate, sodium sulfate and the like.
  • magnesium salts include magnesium sulfate, magnesium nitrate, magnesium chloride and the like.
  • zinc salts include zinc sulfate, zinc nitrate, zinc chloride and the like.
  • the concentration of sulfate in the medium is preferably 0.01 to 0.5% (w / v), more preferably 0.02 to 0.2% (w / v).
  • concentration of magnesium salt in the medium is preferably 0.001 to 0.5% (w / v), more preferably 0.01 to 0.1% (w / v).
  • concentration of zinc salt in the medium is preferably 0.001 to 0.05% (w / v), more preferably 0.005 to 0.05% (w / v).
  • the pH (25 ° C.) of the medium is preferably 3 to 7, more preferably 3.5 to 6.
  • the pH of the medium can be adjusted using a base such as calcium hydroxide, sodium hydroxide, calcium carbonate, or ammonia, or an acid such as sulfuric acid or hydrochloric acid.
  • Preferred examples of the medium include 7.5 to 30% carbon source (of which sorbitol is 10 to 100% by mass), 0.05 to 0.2% ammonium sulfate, and 0.01 to 0.6% potassium dihydrogen phosphate. 0.01-0.1% magnesium sulfate heptahydrate, 0.005-0.05% zinc sulphate heptahydrate, and 3.75-20% calcium carbonate (all concentrations are% (w Liquid medium containing / v)).
  • the sorbitol content in the medium used in the method of the present invention is preferably 1 to 40% (w / v), more preferably 5 to 30% (w / v) in the total amount of the medium.
  • the medium may further contain glucose, and the mass ratio of glucose: sorbitol is preferably 50:50 to 0: 100.
  • the Rhizopus bacterium is cultured in a fumaric acid-producing medium containing the sorbitol, and the fumaric acid is produced by the bacterium.
  • the culture conditions may be the same as those for normal Rhizopus sp.
  • the amount of medium can be about 20 to 80 mL for a 200 mL Erlenmeyer flask, about 50 to 200 mL for a 500 mL Erlenmeyer flask, and about 10 L to 15 L for a 30 L jar fermenter. What is necessary is just to adjust suitably according to a container.
  • the inoculum of the Rhizopus bacterium to the medium may be preferably 10 g to 90 g-bacteria / 100 mL-medium, more preferably 15 g-50 g-bacteria / 100 mL-medium as a wet weight.
  • the culture is carried out at a temperature of 25 to 45 ° C. with stirring at 100 to 300 rpm, preferably 150 to 230 rpm, for 12 hours to 240 hours, preferably 24 hours to 120 hours.
  • aeration is preferably performed at 0.05 to 2 vvm, more preferably 0.1 to 1.5 vvm.
  • Rhizopus sp When inoculating Rhizopus sp. On a medium, a medium having the above-mentioned predetermined sorbitol concentration may be prepared, and then Rhizopus sp. May be inoculated on the medium. Sorbitol and other components necessary for culture may be added so that the concentration of
  • the Rhizopus bacterium cultured in the medium containing sorbitol has improved fumaric acid producing ability. Therefore, according to the method for producing fumaric acid of the present invention, fumaric acid can be produced at a higher yield than the conventional method for producing fumaric acid using Rhizopus sp. Furthermore, Rhizopus sp. Cultivated in a medium containing sorbitol has a reduced ability to produce ethanol, which is a byproduct of fumaric acid production. Therefore, according to the method for producing fumaric acid of the present invention, it is possible to produce fumaric acid more efficiently with less by-product contamination compared to the conventional method.
  • Fumaric acid is recovered from the Rhizopus sp. Culture cultured in the above procedure. For example, after removing the cells from the culture by a gradient method, filtration, centrifugation, etc., and concentrating the obtained culture as necessary, methods such as crystallization method, ion exchange method, solvent extraction method, Alternatively, the fumaric acid in the culture solution can be recovered by applying to these combinations. The recovered fumarate may be further purified as necessary.
  • the Rhizopus sp. Isolated from the culture can be reused for fumaric acid production.
  • the above-described fumaric acid-producing medium can be newly added to the bacteria isolated from the culture, and cultured again under the above conditions.
  • the culture of Rhizopus sp. And the recovery of fumaric acid according to the present invention may be performed by any of batch, semi-batch and continuous methods.
  • Exemplary Embodiment also includes the following substances, production methods, uses or methods as exemplary embodiments. However, the present invention is not limited to these embodiments.
  • a method for producing fumaric acid comprising culturing Rhizopus sp. In a medium containing sorbitol.
  • ⁇ 2> A method for improving the fumaric acid-producing ability of Rhizopus sp., Comprising culturing Rhizopus sp. In a medium containing sorbitol.
  • ⁇ 3> A method for reducing ethanol production ability of Rhizopus sp., Comprising culturing Rhizopus sp. In a medium containing sorbitol.
  • the amount of sorbitol contained in the medium is preferably 10% by mass or more, more preferably 10 to 100% by mass, and further preferably 50 to 100% by mass in the total amount of the carbon source contained in the medium. ⁇ 1> to ⁇ 3>.
  • the amount of the carbon source in the medium is Preferably it is 1% (w / v) or more, more preferably 5% (w / v) or more, more preferably 7.5% (w / v) or more, and preferably 40% (w / v). Or less, more preferably 30% (w / v), or Preferably 1 to 40% (w / v), more preferably 5 to 40% (w / v), still more preferably 7.5 to 40% (w / v), still more preferably 1 to 30% (w / v) v), more preferably 5-30% (w / v), still more preferably 7.5-30% (w / v), ⁇ 4> The method described.
  • the carbon source is a saccharide
  • the amount of the sorbitol is preferably 10% by mass or more, more preferably 10 to 100% by mass, and still more preferably 50 to 100% by mass in the total amount of the saccharide.
  • ⁇ 7> The method according to ⁇ 6>, wherein the saccharide is preferably a sugar and / or a sugar alcohol.
  • the saccharide contains sorbitol and contains a saccharide other than sorbitol, preferably at least one selected from the group consisting of glucose, maltose, starch hydrolysate, fructose, xylose, xylitol and sucrose ⁇ 7>
  • the content of the sorbitol in the medium is preferably 1 to 40% (w / v), more preferably 5 to 30% (w / v) in the total amount of the medium. ⁇ 1> to ⁇ 8. The method according to any one of 8>.
  • the Rhizopus genus is selected from the group Rhizopus delemar, Rhizopus oryzae, Rhizopus arrizus, Rhizopus chinensis, Rhizopus nigricans, Rhizopisti.
  • Rhizopus bacterium is preferably in the form of a pellet or immobilized on a carrier.
  • ⁇ 12> The method according to any one of ⁇ 1> to ⁇ 11>, wherein the culture time is preferably 12 to 240 hours.
  • ⁇ 13> Preferably, the method according to any one of ⁇ 1> and ⁇ 4> to ⁇ 12>, further comprising recovering fumaric acid from the culture obtained in the above culture.
  • Example 1 Production of fumaric acid by culturing Rhizopus (1) Preparation of Rhizopus genus Rhizopus delmar JCM5557 pellets were prepared. Pellets were prepared from the spore suspension with reference to Production Examples 1 to 3 of International Publication No. 2013/157398.
  • Rhizopus spp. Inorganic culture solution for evaluation of fumaric acid production in which 6.0 g of Rhizopus spp. Wet cells obtained in (1) were subjected to a 200 mL Erlenmeyer flask (composition: 10.0%) Carbon source, 0.1% ammonium sulfate, 0.06% potassium dihydrogen phosphate, 0.025% magnesium sulfate heptahydrate, 0.009% zinc sulfate heptahydrate, 5.0% calcium carbonate, In any case, the concentration was inoculated to% (w / v)) and cultured with stirring at 35 ° C. and 170 rpm.
  • glucose, sorbitol, or a mixture thereof shown in Table 1 was used as the carbon source of the culture solution. Immediately after inoculation (0.5 hours) and 120 hours later, the culture supernatant containing no bacterial cells was collected, and fumaric acid, malic acid, succinic acid, and ethanol were collected by the procedure described in Reference Example 1 described later. Was quantified. The results are shown in Table 1. Compared to a medium using only glucose as a carbon source, the Rhizopus culture added with sorbitol had improved fumaric acid productivity and decreased ethanol production.
  • ICSep ICE-ION-300 Guard Column Cartridge (4.0 mm ID ⁇ 2.0 cm, manufactured by TRANSGENOMIC) connected to the analytical column is a polymer column for organic acid analysis ICSep ICE-ION-300 (7.8 mm I.D.). D. ⁇ 30 cm, manufactured by TRANSGENOMC) was used, and the eluent was eluted under conditions of 10 mM sulfuric acid, a flow rate of 0.5 mL / min, and a column temperature of 50 ° C. Each organic acid and ethanol were detected using a suggested refractive index detector (RI detector) and a UV detector (detection wavelength 210 nm), respectively.
  • RI detector refractive index detector
  • UV detector detection wavelength 210 nm
  • Concentration calibration curves consist of standard samples [fumaric acid (sales code 063-00655, manufactured by Wako Pure Chemical Industries), malic acid (sales code 135-00562, manufactured by Wako Pure Chemical Industries), succinic acid (sales code 194- 04335, manufactured by Wako Pure Chemical Industries, Ltd.) and ethanol (product number 057-00456, manufactured by Wako Pure Chemical Industries, Ltd.)]. Based on each concentration calibration curve, each component was quantified.

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Abstract

Provided is an efficient microbiological production method for fumaric acid. A method for producing fumaric acid includes culturing Rhizopus spp. in a medium containing sorbitol.

Description

フマル酸の製造方法Process for producing fumaric acid
　本発明は、フマル酸の製造方法に関する。 The present invention relates to a method for producing fumaric acid.
　フマル酸は、酸味料や抗菌剤としてよく用いられているほか、リンゴ酸やアスパラギン酸等の中間物質として食品製造に使用されたり、合成樹脂や生分解性ポリマーの原料として用いられるなど、工業的な価値が高い物質である。フマル酸は、工業的には化学合成法やリゾプス属菌（Ｒｈｉｚｏｐｕｓ）等の発酵菌を用いて製造されてきた。従前は、より低コストなため化学合成法が主流であったが、近年、原料の高騰や、食品用途の際の安全性、環境負荷などの観点から、微生物学的方法が再び注目されている。 Fumaric acid is often used as an acidulant and antibacterial agent, and is also used in food production as an intermediate substance such as malic acid and aspartic acid. It is also used as a raw material for synthetic resins and biodegradable polymers. It is a highly valuable substance. Industrially, fumaric acid has been produced using chemical synthesis methods and fermenting bacteria such as Rhizopus. Previously, chemical synthesis methods were the mainstream because of their lower cost, but in recent years, microbiological methods have attracted attention again from the viewpoints of soaring raw materials, safety during food use, environmental impact, etc. .
　フマル酸は、ＴＣＡサイクルの中間物質として知られているが、リゾプス属菌においては、主にグルコースから生じたピルビン酸から始まる還元的ＴＣＡ経路において生成されると考えられている。一方で、リゾプス属菌中にはピルビン酸からエタノールを生成する経路も存在するため、リゾプス属菌によるフマル酸生産には、副産物としてエタノールが混入するという問題がある。 Fumaric acid is known as an intermediate substance in the TCA cycle, but in Rhizopus sp., It is considered that it is produced in a reductive TCA pathway starting from pyruvic acid generated mainly from glucose. On the other hand, in Rhizopus sp., There is a pathway for producing ethanol from pyruvic acid. Therefore, the production of fumaric acid by Rhizopus sp has a problem that ethanol is mixed as a by-product.
　フマル酸生産量が多く、エタノール産生の少ない変異リゾプス属菌が報告されている（非特許文献１）。しかし、リゾプス属菌の遺伝学的背景は未だ十分に研究されていないことから、遺伝子組換え技術によるフマル酸高生産性リゾプス属菌の開発は容易ではない。 Mutant Rhizopus spp. With high fumaric acid production and low ethanol production have been reported (Non-patent Document 1). However, since the genetic background of Rhizopus spp. Has not yet been fully studied, it is not easy to develop a high fumarate-producing Rhizopus spp.
　グルコース以外の炭素源を用いた有機酸の微生物学的生産方法が報告されている。特許文献１には、脂肪酸エステルやトリグリセリドを添加した培地でリゾプス属菌を培養する有機酸の製造方法が開示されている。特許文献２には、グルコース以外の種々の糖を添加した培地でＡｓｐｅｒｇｉｌｌｕｓ属菌を培養し、リンゴ酸生産性を調べたことが開示されている。しかし、上記グルコース以外の糖を用いた場合のリンゴ酸生産性は、グルコースと比べて低かった。特許文献３には、バイオマスから生産した粗ソルビトールから、Ａｃｔｉｎｏｂａｃｉｌｌｕｓ　ｓｕｃｃｉｎｏｇｅｎｅｓ等の種々の菌にコハク酸を生産させたことが開示されている。特許文献４には、組換えＡｃｔｉｎｏｂａｃｉｌｌｕｓ　ｓｕｃｃｉｎｏｇｅｎｅｓにソルビトールからコハク酸を生産させたことが開示されている。非特許文献２には、ソルビトール添加培地で組換え大腸菌を培養することにより、グルコール添加培地に比べてコハク酸生産性が向上したが、同時にエタノール生産量も増加したことが開示されている。
　　（特許文献１）米国特許第４５６４５９４号公報
　　（特許文献２）英国特許第８８４０２９号公報
　　（特許文献３）米国特許公開公報第２０１４／００９３９２６号
　　（特許文献４）米国特許公開公報第２０１４／００９３９２５号
　　（非特許文献１）Korean J Chem Eng, 2010, 27(1):183-186
　　（非特許文献２）J Ind Microbiol Biotechnol, 2005, 32(3):87-93 A method for microbiological production of organic acids using carbon sources other than glucose has been reported. Patent Document 1 discloses a method for producing an organic acid in which Rhizopus is cultured in a medium to which a fatty acid ester or triglyceride is added. Patent Document 2 discloses that Aspergillus genus bacteria were cultured in a medium to which various sugars other than glucose were added, and malic acid productivity was examined. However, the malic acid productivity in the case of using sugars other than glucose was lower than that of glucose. Patent Document 3 discloses that succinic acid was produced by various bacteria such as Actinobacillus succinogenes from crude sorbitol produced from biomass. Patent Document 4 discloses that succinic acid was produced from sorbitol in recombinant Actinobacillus succinogenes. Non-Patent Document 2 discloses that succinic acid productivity was improved by culturing recombinant Escherichia coli in a sorbitol-added medium as compared to a glucose-added medium, but at the same time, ethanol production was also increased.
(Patent Literature 1) US Pat. No. 4,564,594 (Patent Literature 2) British Patent No. 884029 (Patent Literature 3) US Patent Publication No. 2014/0093926 (Patent Literature 4) US Patent Publication No. 2014/0093925 (Non-Patent Document 1) Korean J Chem Eng, 2010, 27 (1): 183-186
(Non-patent document 2) J Ind Microbiol Biotechnol, 2005, 32 (3): 87-93
　本発明は、ソルビトールを含有する培地でリゾプス属菌を培養することを含む、フマル酸の製造方法を提供する。 The present invention provides a method for producing fumaric acid, comprising culturing Rhizopus sp. In a medium containing sorbitol.
発明の詳細な説明Detailed Description of the Invention
　本発明は、効率のよいフマル酸の微生物学的生産方法に関する。 The present invention relates to an efficient microbiological production method of fumaric acid.
　本発明者らは、微生物のフマル酸生産性を向上させるべく種々検討した結果、炭素源にソルビトールを含む培地でリゾプス属菌を培養することによって、当該菌のフマル酸生産性が向上し、かつ副産物であるエタノールの生成が抑制されることを見出した。 As a result of various studies to improve the fumaric acid productivity of microorganisms, the present inventors have improved the fumaric acid productivity of the fungus by culturing Rhizopus sp. In a medium containing sorbitol as a carbon source, and It has been found that the production of ethanol as a by-product is suppressed.
　本発明の方法によれば、従来のリゾプス属菌によるフマル酸製造法と比べて、フマル酸の収量を増加させることができ、かつ副産物であるエタノールの生成を抑えることができる。したがって、本発明によれば、より効率よくかつ低コストなフマル酸製造が可能になる。 According to the method of the present invention, the yield of fumaric acid can be increased and the production of ethanol as a byproduct can be suppressed as compared with the conventional method of producing fumaric acid by Rhizopus sp. Therefore, according to the present invention, fumaric acid can be produced more efficiently and at low cost.
　本発明のフマル酸の製造方法においては、ソルビトールを含有する培地で、リゾプス属菌を培養する。培養終了後、生産されたフマル酸を培地から回収する。 In the method for producing fumaric acid of the present invention, Rhizopus is cultured in a medium containing sorbitol. After completion of the culture, the produced fumaric acid is recovered from the medium.
（１．リゾプス属菌）
（１．１．菌種）
　本発明のフマル酸の製造方法に用いられるリゾプス属菌の種類としては、特に限定されないが、例えば、Ｒｈｉｚｏｐｕｓ　ｄｅｌｅｍａｒ、Ｒｈｉｚｏｐｕｓ　ｏｒｙｚａｅ、Ｒｈｉｚｏｐｕｓ　ａｒｒｈｉｚｕｓ、Ｒｈｉｚｏｐｕｓ　ｃｈｉｎｅｎｓｉｓ、Ｒｈｉｚｏｐｕｓ　ｎｉｇｒｉｃａｎｓ、Ｒｈｉｚｏｐｕｓ　ｔｏｎｋｉｎｅｎｓｉｓ、Ｒｈｉｚｏｐｕｓ　ｔｒｉｔｉｃｉなどが挙げられる。これらのリゾプス属菌種は、単独で使用すればよいが、２種以上組み合わせて使用することもできる。 (1. Rhizopus sp.)
(1.1. Bacterial species)
The type of Rhizopus genus used in the method for producing fumaric acid of the present invention is not particularly limited, and examples thereof include Rhizopus delemar, Rhizopus oryzae, Rhizopus ariziz, Rhizopus chinhis, . These Rhizopus species may be used alone or in combination of two or more.
（１．２．リゾプス属菌の調製）
　本発明の方法において、上記リゾプス属菌は、ペレットの形態で用いることが好ましい。あるいは、本発明の方法において、上記リゾプス属菌は、担体に固定化されたリゾプス属菌であってもよく、または該ペレットと該担体に固定化された菌とが併用されてもよい。本明細書において、リゾプス属菌の「ペレット」とは、液体培養により菌糸が自発的に形成した数百μｍ～数ｍｍの大きさの菌糸塊をいい、「担体に固定化されたリゾプス属菌」とは、担体に保持又は包埋されたリゾプス属菌をいう。上記リゾプス属菌のペレット及び担体に固定化されたリゾプス属菌は、商業的に入手したものを使用しても、胞子又は菌糸から調製したものを使用してもよい。 (1.2. Preparation of Rhizopus sp.)
In the method of the present invention, the Rhizopus bacterium is preferably used in the form of a pellet. Alternatively, in the method of the present invention, the Rhizopus bacterium may be a Rhizopus bacterium immobilized on a carrier, or the pellet and the bacterium immobilized on the carrier may be used in combination. In the present specification, a “pellet” of Rhizopus sp. Means a hypha mass having a size of several hundred μm to several mm spontaneously formed by hyphae by liquid culture, and “Rhizopus sp. "Means Rhizopus spp. Held or embedded in a carrier. The Rhizopus genus bacteria immobilized on the Rhizopus bacterium pellets and the carrier may be those obtained commercially or those prepared from spores or hyphae.
（１．２．１．ペレットの調製）
　リゾプス属菌ペレットは、公知の方法で調製することができる。例えば、リゾプス属菌の胞子懸濁液を調製し（工程Ａ）、それを培養液で培養して胞子を発芽させて菌糸体を調製し（工程Ｂ）、好適には、さらに工程Ｂで得られた菌糸体を増殖させて（工程Ｃ）、ペレットを形成させる。調製されたペレットは、ろ別、遠心分離等により培養液から分離回収し、フマル酸製造に使用することができる。工程Ａ～Ｃの好ましい実施形態を以下に詳述する。 (1.2.1. Preparation of pellets)
Rhizopus pellets can be prepared by a known method. For example, a spore suspension of Rhizopus spp. Is prepared (Step A), and it is cultured in a culture solution to germinate spores to prepare a mycelium (Step B), preferably obtained in Step B. The obtained mycelium is grown (step C) to form a pellet. The prepared pellet can be separated and recovered from the culture solution by filtration, centrifugation, etc., and used for fumaric acid production. A preferred embodiment of steps A to C is described in detail below.
＜工程Ａ：胞子懸濁液の調製＞
　リゾプス属菌の胞子を、例えば、無機寒天培地（組成例：２％グルコース、０．１％硫酸アンモニウム、０．０６％リン酸２水素カリウム、０．０２５％硫酸マグネシウム・７水和物、０．００９％硫酸亜鉛・７水和物、１．５％寒天、いずれも濃度は％（ｗ／ｖ））、ＰＤＡ培地、等の培地に接種し、１０～４０℃、好ましくは２７～３０℃にて、７～１０日間静置培養を行い、次いで生理食塩水などに懸濁することで、胞子懸濁液を調製することができる。胞子懸濁液には菌糸体が含まれていても、含まれていなくてもよい。 <Step A: Preparation of spore suspension>
The spores of Rhizopus sp., For example, an inorganic agar medium (composition example: 2% glucose, 0.1% ammonium sulfate, 0.06% potassium dihydrogen phosphate, 0.025% magnesium sulfate heptahydrate, 0. Inoculate 009% zinc sulfate heptahydrate, 1.5% agar (both concentrations are% (w / v)), PDA medium, etc., at 10-40 ° C, preferably 27-30 ° C Then, the spore suspension can be prepared by performing stationary culture for 7 to 10 days and then suspending in physiological saline or the like. The spore suspension may or may not contain mycelium.
＜工程Ｂ：菌糸体の調製＞
　上記で得られた胞子懸濁液を、培養液に接種して培養し、胞子を発芽させて菌糸体を得る。当該培養は、通常の手順にて行なえばよい。培養液に接種する糸状菌の胞子数は、１×１０2～１×１０8個－胞子／ｍＬ－培養液、好ましくは１×１０2～５×１０4個－胞子／ｍＬ－培養液、より好ましくは５×１０2～１×１０4個－胞子／ｍＬ－培養液、さらに好ましくは１×１０3～１×１０4個－胞子／ｍＬ－培養液である。培養液には、市販の培地、例えば、ポテトデキストロース培地（以下、ＰＤＢ培地という場合もある。例えばベクトン・ディッキンソン　アンド　カンパニー製）、Ｌｕｒｉａ－Ｂｅｒｔａｎｉ培地（以下、ＬＢ培地という場合もある。例えば日本製薬社製）、Ｎｕｔｒｉｅｎｔ　Ｂｒｏｔｈ（以下、ＮＢ培地という場合もある。例えばベクトン・ディッキンソン　アンド　カンパニー製）、Ｓａｂｏｕｒａｕｄ培地（以下、ＳＢ培地という場合もある、例えばＯＸＯＩＤ社製）等が利用できる。該培養液には、発芽率と菌体生育の観点から、炭素源としてグルコース、キシロースなどの単糖、シュークロース、ラクトース、マルトースなどのオリゴ糖、又はデンプン等の多糖；グリセリン、クエン酸などの生体成分；窒素源として硫酸アンモニウム、尿素等；その他無機物としてナトリウム、カリウム、マグネシウム、亜鉛、鉄、リン酸等の各種塩類、を適宜添加することができる。単糖、オリゴ糖、多糖及びグリセリンの好ましい濃度は０．１～３０％（ｗ／ｖ）、クエン酸の好ましい濃度は０．０１～１０％（ｗ／ｖ）、窒素源の好ましい濃度は０．０１～１％（ｗ／ｖ）、無機物の好ましい濃度は０．０００１～０．５％（ｗ／ｖ）である。上記培養液に胞子懸濁液を接種し、好ましくは８０～２５０ｒｐｍ、より好ましくは１００～１７０ｒｐｍで攪拌しながら、２５～４２．５℃の培養温度制御下で、好ましくは２４～１２０時間、より好ましくは４８～７２時間培養する。培養に供する培養液の量は、培養容器にあわせて適宜調整すればよいが、例えば、２００ｍＬ容バッフル付フラスコの場合は５０～１００ｍＬ程度、５００ｍＬ容バッフル付フラスコの場合は１００～３００ｍＬ程度であればよい。この培養により、接種した胞子は発芽し、菌糸体へと成長する。 <Step B: Preparation of mycelium>
The spore suspension obtained above is inoculated into a culture solution and cultured, and the spores are germinated to obtain mycelium. The culture may be performed according to a normal procedure. The number of spores of the filamentous fungus inoculated into the culture solution is 1 × 10 2 to 1 × 10 8 spore / mL-culture solution, preferably 1 × 10 2 to 5 × 10 4 spore / mL-culture solution, More preferred is 5 × 10 2 to 1 × 10 4 spores / mL-culture solution, and further preferred is 1 × 10 3 to 1 × 10 4 spores / mL-culture solution. The culture solution may be a commercially available medium, for example, potato dextrose medium (hereinafter also referred to as PDB medium, for example, Becton Dickinson and Company), Luria-Bertani medium (hereinafter also referred to as LB medium, for example, Nippon Pharmaceutical). For example, Betroton Dickinson and Company), Sabouraud medium (hereinafter also referred to as SB medium, for example, manufactured by OXOID), and the like can be used. From the viewpoints of germination rate and cell growth, the culture solution may be a monosaccharide such as glucose or xylose as a carbon source, an oligosaccharide such as sucrose, lactose or maltose, or a polysaccharide such as starch; glycerin, citric acid or the like. Biological components: ammonium sulfate, urea, etc. as a nitrogen source; various salts such as sodium, potassium, magnesium, zinc, iron, phosphoric acid, etc. can be added as appropriate. The preferred concentration of monosaccharide, oligosaccharide, polysaccharide and glycerin is 0.1-30% (w / v), the preferred concentration of citric acid is 0.01-10% (w / v), and the preferred concentration of nitrogen source is 0 .01 to 1% (w / v), and the preferred concentration of the inorganic substance is 0.0001 to 0.5% (w / v). The culture broth is inoculated with a spore suspension, and is preferably stirred at 80 to 250 rpm, more preferably 100 to 170 rpm, under a culture temperature control of 25 to 42.5 ° C., preferably for 24 to 120 hours. The culture is preferably performed for 48 to 72 hours. The amount of the culture solution used for the culture may be appropriately adjusted according to the culture vessel. For example, it may be about 50 to 100 mL for a 200 mL baffled flask and about 100 to 300 mL for a 500 mL baffled flask. That's fine. By this culture, the inoculated spores germinate and grow into mycelium.
＜工程Ｃ：菌糸体の増殖＞
　工程Ｃは、工程Ｂで得られた菌糸体をさらに培養して増殖させる工程である。本工程は必須ではないが、フマル酸の生産性向上の観点からは、本工程を行うことが好ましい。工程Ｃで使用する増殖用の培養液は特に限定されないが、通常使用されるグルコースを含む無機培養液であればよく、例えば、７．５～３０％グルコース、０．０５～２％硫酸アンモニウム、０．０３～０．６％リン酸２水素カリウム、０．０１～０．１％硫酸マグネシウム・７水和物、０．００５～０．０５％硫酸亜鉛・７水和物、及び３．７５～２０％炭酸カルシウム（いずれも濃度は％（ｗ／ｖ））を含有する培養液等が挙げられる。当該培養液の量は、培養容器にあわせて適宜調整すればよいが、例えば、５００ｍＬ容三角フラスコの場合は５０～３００ｍＬ、好ましくは１００～２００ｍＬであればよい。この培養液に、工程Ｂで培養した菌体を、湿重量として１～６ｇ－菌体／１００ｍＬ－培養液、好ましくは３～４ｇ－菌体／１００ｍＬ－培養液となるよう接種し、１００～３００ｒｐｍ、好ましくは１７０～２３０ｒｐｍで攪拌しながら、２５～４２．５℃の培養温度制御下で、１２～１２０時間、好ましくは２４～７２時間培養する。 <Process C: Mycelium growth>
Step C is a step of further culturing and growing the mycelium obtained in Step B. Although this step is not essential, it is preferable to perform this step from the viewpoint of improving the fumaric acid productivity. The growth medium used in Step C is not particularly limited, and may be any inorganic culture medium containing glucose that is usually used. For example, 7.5 to 30% glucose, 0.05 to 2% ammonium sulfate, 0 0.03-0.6% potassium dihydrogen phosphate, 0.01-0.1% magnesium sulfate heptahydrate, 0.005-0.05% zinc sulfate heptahydrate, and 3.75- Examples include a culture solution containing 20% calcium carbonate (both concentrations are% (w / v)). The amount of the culture solution may be appropriately adjusted according to the culture vessel. For example, in the case of a 500 mL Erlenmeyer flask, it may be 50 to 300 mL, preferably 100 to 200 mL. To this culture broth, the cells cultured in step B are inoculated in a wet weight of 1 to 6 g-cells / 100 mL-culture solution, preferably 3 to 4 g-cells / 100 mL-culture solution, and 100 to 100- The culture is performed for 12 to 120 hours, preferably 24 to 72 hours under a culture temperature control of 25 to 42.5 ° C. while stirring at 300 rpm, preferably 170 to 230 rpm.
（１．２．２．固定化リゾプス属菌の調製）
　担体に固定化されたリゾプス属菌は、公知の方法で調製することができる。例えば、リゾプス属菌の胞子を、担体の存在する液体培地に植菌後、培養し、胞子から発芽した菌糸が取り込まれた担体を回収する（特開２０１３－２４０３２１号公報参照）。リゾプス属菌固定化用の担体の材質としては、ウレタン系重合体、オレフィン系重合体、ジエン系重合体、縮合系重合体、シリコーン系重合体、フッ素系重合体等が挙げられ、その構造は、発泡体、薄片体、シート体、中空体、樹脂成型体等が好ましい。該担体の形状は、平板状、多層板状、波板状、四面体状、球状、紐状、網状、円柱状、格子状、円筒状等のいずれでもよく、そのサイズは、最長辺の長さで０．１ｍｍ～１０ｍｍが好ましく、０．３～５ｍｍがより好ましく、０．５～２ｍｍがさらに好ましい。リゾプス属菌固定化担体は、ろ別、遠心分離等により培養液から分離回収し、フマル酸製造に使用することができる。 (1.2.2. Preparation of immobilized Rhizopus sp.)
The Rhizopus bacterium immobilized on the carrier can be prepared by a known method. For example, the spores of Rhizopus sp. Are inoculated into a liquid medium containing a carrier and cultured, and the carrier in which the mycelium germinated from the spore is taken is recovered (see JP 2013-240321 A). Examples of the material of the carrier for immobilizing Rhizopus sp. Include urethane polymers, olefin polymers, diene polymers, condensation polymers, silicone polymers, fluorine polymers, and the structure thereof. A foam, a thin piece, a sheet, a hollow body, a resin molded body and the like are preferable. The shape of the carrier may be any of a flat plate shape, a multilayer plate shape, a corrugated plate shape, a tetrahedron shape, a spherical shape, a string shape, a net shape, a columnar shape, a lattice shape, a cylindrical shape, and the size is the length of the longest side. The thickness is preferably 0.1 mm to 10 mm, more preferably 0.3 to 5 mm, and even more preferably 0.5 to 2 mm. The Rhizopus bacterium-immobilized carrier can be separated and recovered from the culture solution by filtration, centrifugation, or the like, and used for fumaric acid production.
（２．フマル酸生産用培地）
　本発明のフマル酸製造方法に使用される培地としては、炭素源にソルビトールを含む以外は、リゾプス属菌による有機酸生産のために通常用いられるものを使用すればよい。通常、培地は液体培地であり、合成培地、天然培地、及び合成培地に天然成分を添加した半合成培地のいずれであってもよい。当該培地には、炭素源、窒素源、無機塩等が含まれるのが一般的であるが、各成分組成は、上述した炭素源以外は、適宜選択可能である。以下に好ましい培地組成について詳述する。以下に記載する培地中の各成分の濃度は、初発（培地調製時又は培養開始時）の濃度を表す。 (2. Fumaric acid production medium)
As a medium used for the fumaric acid production method of the present invention, a medium usually used for organic acid production by Rhizopus sp. May be used except that the carbon source contains sorbitol. Usually, the medium is a liquid medium, and may be any of a synthetic medium, a natural medium, and a semi-synthetic medium obtained by adding a natural component to a synthetic medium. The medium generally contains a carbon source, a nitrogen source, an inorganic salt, and the like, but each component composition can be appropriately selected except for the above-described carbon source. The preferable medium composition is described in detail below. The density | concentration of each component in the culture medium described below represents the density | concentration of the first time (at the time of culture-medium preparation or culture | cultivation start).
　上記培地は、ソルビトールを含有する。該培地に含まれるソルビトールの量は、培地に含まれる炭素源の全量中、好ましくは１０質量％以上、より好ましくは１０～１００質量％、さらに好ましくは５０～１００質量％である。好適には、該培地の炭素源は、糖、糖アルコールなどの糖類であり、該糖類の全量中に、好ましくは１０質量％以上、より好ましくは１０～１００質量％、さらに好ましくは５０～１００質量％のソルビトールが含まれる。上記培地は、上記糖類以外の炭素源、例えば、脂肪酸エステルやトリグリセリドを含んでいてもよいが、その場合も、該培地中のソルビトールの量は、該糖類中に、好ましくは１０質量％以上、より好ましくは１０～１００質量％、さらに好ましくは５０～１００質量％である。培地にグルコースを含む場合、グルコース／（ソルビトール＋グルコース）は、質量比で、好ましくは０以上０．５以下、より好ましくは０以上０．３以下、さらに好ましくは０以上０．１以下である。 The above medium contains sorbitol. The amount of sorbitol contained in the medium is preferably 10% by mass or more, more preferably 10 to 100% by mass, and still more preferably 50 to 100% by mass in the total amount of the carbon source contained in the medium. Suitably, the carbon source of the medium is a saccharide such as sugar or sugar alcohol, and is preferably 10% by mass or more, more preferably 10 to 100% by mass, and still more preferably 50 to 100% in the total amount of the saccharide. Mass% sorbitol is included. The medium may contain a carbon source other than the saccharide, for example, a fatty acid ester or a triglyceride. In this case, the amount of sorbitol in the medium is preferably 10% by mass or more in the saccharide. More preferably, it is 10 to 100% by mass, and still more preferably 50 to 100% by mass. When glucose is contained in the medium, glucose / (sorbitol + glucose) is preferably in the range of 0 to 0.5, more preferably 0 to 0.3, and even more preferably 0 to 0.1. .
　上記培地の炭素源である糖類は、ソルビトール以外の糖類を含んでいてもよい。該ソルビトール以外の糖類の例としては、グルコース、マルトース、でんぷん加水分解物、フルクトース、キシロース、キシリトール、スクロース等が挙げられ、このうち、グルコース、フルクトース及びキシリトールが好ましい。これらの糖類は、単独で又は２種以上組み合わせて使用することができる。 The saccharide that is the carbon source of the medium may contain a saccharide other than sorbitol. Examples of saccharides other than sorbitol include glucose, maltose, starch hydrolysate, fructose, xylose, xylitol, sucrose and the like, among which glucose, fructose and xylitol are preferred. These saccharides can be used alone or in combination of two or more.
　上記培地中の炭素源（好ましくはソルビトールを含む糖類）の濃度は、好ましくは１％（ｗ／ｖ）以上、より好ましくは５％（ｗ／ｖ）以上であって、かつ好ましくは４０％（ｗ／ｖ）以下、より好ましくは３０％（ｗ／ｖ）以下である。あるいは、上記培地中の炭素源（ソルビトールを含む）の濃度は、好ましくは１～４０％（ｗ／ｖ）、より好ましくは５～３０％（ｗ／ｖ）である。 The concentration of the carbon source (preferably saccharide containing sorbitol) in the medium is preferably 1% (w / v) or more, more preferably 5% (w / v) or more, and preferably 40% ( w / v) or less, more preferably 30% (w / v) or less. Alternatively, the concentration of the carbon source (including sorbitol) in the medium is preferably 1 to 40% (w / v), more preferably 5 to 30% (w / v).
　上記培地中の窒素源の例としては、尿素、硝酸アンモニウム、硝酸カリウム、硝酸ナトリウム等の含窒素化合物が挙げられる。該培地中の窒素源の濃度は、好ましくは０．００１～０．５％（ｗ／ｖ）、より好ましくは０．０５～０．２％（ｗ／ｖ）である。 Examples of the nitrogen source in the medium include nitrogen-containing compounds such as urea, ammonium nitrate, potassium nitrate, and sodium nitrate. The concentration of the nitrogen source in the medium is preferably 0.001 to 0.5% (w / v), more preferably 0.05 to 0.2% (w / v).
　上記培地には、硫酸塩、マグネシウム塩、亜鉛塩などを含有することができる。硫酸塩の例としては、硫酸マグネシウム、硫酸亜鉛、硫酸カリウム、硫酸ナトリウム等が挙げられる。マグネシウム塩の例としては、硫酸マグネシウム、硝酸マグネシウム、塩化マグネシウム等が挙げられる。亜鉛塩の例としては、具体的には、硫酸亜鉛、硝酸亜鉛、塩化亜鉛等が挙げられる。該培地中の硫酸塩の濃度は、好ましくは０．０１～０．５％（ｗ／ｖ）、より好ましくは０．０２～０．２％（ｗ／ｖ）である。該培地中のマグネシウム塩の濃度は、好ましくは０．００１～０．５％（ｗ／ｖ）、より好ましくは０．０１～０．１％（ｗ／ｖ）である。該培地中の亜鉛塩の濃度は、好ましくは０．００１～０．０５％（ｗ／ｖ）、より好ましくは０．００５～０．０５％（ｗ／ｖ）である。 The above medium can contain sulfate, magnesium salt, zinc salt and the like. Examples of sulfates include magnesium sulfate, zinc sulfate, potassium sulfate, sodium sulfate and the like. Examples of magnesium salts include magnesium sulfate, magnesium nitrate, magnesium chloride and the like. Specific examples of zinc salts include zinc sulfate, zinc nitrate, zinc chloride and the like. The concentration of sulfate in the medium is preferably 0.01 to 0.5% (w / v), more preferably 0.02 to 0.2% (w / v). The concentration of magnesium salt in the medium is preferably 0.001 to 0.5% (w / v), more preferably 0.01 to 0.1% (w / v). The concentration of zinc salt in the medium is preferably 0.001 to 0.05% (w / v), more preferably 0.005 to 0.05% (w / v).
　上記培地のｐＨ（２５℃）は、好ましくは３～７、より好ましくは３．５～６である。培地のｐＨは、水酸化カルシウム、水酸化ナトリウム、炭酸カルシウム、アンモニア等の塩基、又は硫酸、塩酸等の酸を用いて調整することができる。 The pH (25 ° C.) of the medium is preferably 3 to 7, more preferably 3.5 to 6. The pH of the medium can be adjusted using a base such as calcium hydroxide, sodium hydroxide, calcium carbonate, or ammonia, or an acid such as sulfuric acid or hydrochloric acid.
　上記培地の好ましい例としては、７．５～３０％炭素源（うち、ソルビトール１０～１００質量％）、０．０５～０．２％硫酸アンモニウム、０．０１～０．６％リン酸２水素カリウム、０．０１～０．１％硫酸マグネシウム・７水和物、０．００５～０．０５％硫酸亜鉛・７水和物、及び３．７５～２０％炭酸カルシウム（いずれも濃度は％（ｗ／ｖ））を含有する液体培地が挙げられる。 Preferred examples of the medium include 7.5 to 30% carbon source (of which sorbitol is 10 to 100% by mass), 0.05 to 0.2% ammonium sulfate, and 0.01 to 0.6% potassium dihydrogen phosphate. 0.01-0.1% magnesium sulfate heptahydrate, 0.005-0.05% zinc sulphate heptahydrate, and 3.75-20% calcium carbonate (all concentrations are% (w Liquid medium containing / v)).
　あるいは、本発明の方法で使用される培地におけるソルビトールの含有量は、培地全量中、好ましくは１～４０％（ｗ／ｖ）、より好ましくは５～３０％（ｗ／ｖ）であり得る。該培地は、さらにグルコースを含んでいてもよく、該グルコース：ソルビトールの質量比は、好ましくは５０：５０～０：１００である。 Alternatively, the sorbitol content in the medium used in the method of the present invention is preferably 1 to 40% (w / v), more preferably 5 to 30% (w / v) in the total amount of the medium. The medium may further contain glucose, and the mass ratio of glucose: sorbitol is preferably 50:50 to 0: 100.
（３．リゾプス属菌の培養）
（３．１．培養手順）
　本発明のフマル酸の製造方法においては、上記ソルビトールを含有するフマル酸生産用培地で、上記リゾプス属菌を培養し、該菌にフマル酸を生産させる。該培養の条件は、通常のリゾプス属菌の培養条件に従えばよい。例えば、培地の量は、２００ｍＬ容三角フラスコの場合は２０～８０ｍＬ程度、５００ｍＬ容三角フラスコの場合は５０～２００ｍＬ程度、３０Ｌジャーファーメンターの場合は１０Ｌ～１５Ｌ程度とすることができるが、培養容器にあわせて適宜調整すればよい。培地に対する上記リゾプス属菌の接種量は、好ましくは湿重量として１０ｇ～９０ｇ－菌体／１００ｍＬ－培地、より好ましくは１５ｇ～５０ｇ－菌体／１００ｍＬ－培地であり得る。好適には、培養は、１００～３００ｒｐｍ、好ましくは１５０～２３０ｒｐｍで攪拌しながら、２５～４５℃の温度下で、１２時間～２４０時間、好ましくは２４時間～１２０時間行われる。ジャーファーメンターを用いる場合は、通気は好ましくは０．０５～２ｖｖｍ、より好ましくは０．１～１．５ｖｖｍにて行う。 (3. Rhizopus culture)
(3.1. Culture procedure)
In the method for producing fumaric acid of the present invention, the Rhizopus bacterium is cultured in a fumaric acid-producing medium containing the sorbitol, and the fumaric acid is produced by the bacterium. The culture conditions may be the same as those for normal Rhizopus sp. For example, the amount of medium can be about 20 to 80 mL for a 200 mL Erlenmeyer flask, about 50 to 200 mL for a 500 mL Erlenmeyer flask, and about 10 L to 15 L for a 30 L jar fermenter. What is necessary is just to adjust suitably according to a container. The inoculum of the Rhizopus bacterium to the medium may be preferably 10 g to 90 g-bacteria / 100 mL-medium, more preferably 15 g-50 g-bacteria / 100 mL-medium as a wet weight. Suitably, the culture is carried out at a temperature of 25 to 45 ° C. with stirring at 100 to 300 rpm, preferably 150 to 230 rpm, for 12 hours to 240 hours, preferably 24 hours to 120 hours. When a jar fermenter is used, aeration is preferably performed at 0.05 to 2 vvm, more preferably 0.1 to 1.5 vvm.
　培地にリゾプス属菌を接種する場合、上述した所定のソルビトール濃度の培地を作製してから、これにリゾプス属菌を接種してもよいが、リゾプス属菌を含む培地に対して、上述した所定の濃度になるようにソルビトールや、その他の培養に必要な成分を添加してもよい。 When inoculating Rhizopus sp. On a medium, a medium having the above-mentioned predetermined sorbitol concentration may be prepared, and then Rhizopus sp. May be inoculated on the medium. Sorbitol and other components necessary for culture may be added so that the concentration of
（３．２．フマル酸生産能の向上）
　上記手順に従って、上記ソルビトールを含有する培地で培養されたリゾプス属菌は、フマル酸生産能が向上する。したがって、本発明のフマル酸の製造方法によれば、従来のリゾプス属菌を用いたフマル酸製造法と比べて、より高収量でフマル酸を製造することができる。さらに、上記ソルビトールを含有する培地で培養されたリゾプス属菌は、フマル酸製造の副産物であるエタノールの生産能が低減している。したがって、本発明のフマル酸の製造方法によれば、従来法と比べて、副産物の混入が少ない、より効率のよいフマル酸の製造が可能になる。 (3.2. Improvement of fumaric acid production ability)
According to the above procedure, the Rhizopus bacterium cultured in the medium containing sorbitol has improved fumaric acid producing ability. Therefore, according to the method for producing fumaric acid of the present invention, fumaric acid can be produced at a higher yield than the conventional method for producing fumaric acid using Rhizopus sp. Furthermore, Rhizopus sp. Cultivated in a medium containing sorbitol has a reduced ability to produce ethanol, which is a byproduct of fumaric acid production. Therefore, according to the method for producing fumaric acid of the present invention, it is possible to produce fumaric acid more efficiently with less by-product contamination compared to the conventional method.
（４．フマル酸の回収）
　上記の手順で培養したリゾプス属菌培養物から、フマル酸を回収する。例えば、傾斜法、ろ過、遠心分離などにより培養物から菌体を除去し、得られた培養物を、必要に応じて濃縮した後、晶析法、イオン交換法、溶剤抽出法等の方法、又はこれらの組み合わせにかけ、該培養液中のフマル酸を回収することができる。回収したフマル酸塩は、必要に応じてさらに精製されてもよい。 (4. Recovery of fumaric acid)
Fumaric acid is recovered from the Rhizopus sp. Culture cultured in the above procedure. For example, after removing the cells from the culture by a gradient method, filtration, centrifugation, etc., and concentrating the obtained culture as necessary, methods such as crystallization method, ion exchange method, solvent extraction method, Alternatively, the fumaric acid in the culture solution can be recovered by applying to these combinations. The recovered fumarate may be further purified as necessary.
　培養物から分離されたリゾプス属菌は、フマル酸生産に再利用することができる。例えば、培養物から分離した菌に上述したフマル酸生産用培地を新たに加え、再び上記条件で培養することができる。本発明によるリゾプス属菌の培養及びフマル酸の回収は、回分式、半回分式及び連続式のいずれの方法で行ってもよい。 The Rhizopus sp. Isolated from the culture can be reused for fumaric acid production. For example, the above-described fumaric acid-producing medium can be newly added to the bacteria isolated from the culture, and cultured again under the above conditions. The culture of Rhizopus sp. And the recovery of fumaric acid according to the present invention may be performed by any of batch, semi-batch and continuous methods.
（５．例示的実施形態）
　本発明はまた、例示的実施形態として以下の物質、製造方法、用途又は方法を包含する。但し、本発明はこれらの実施形態に限定されない。 5. Exemplary Embodiment
The present invention also includes the following substances, production methods, uses or methods as exemplary embodiments. However, the present invention is not limited to these embodiments.
＜１＞ソルビトールを含有する培地でリゾプス属菌を培養することを含む、フマル酸の製造方法。 <1> A method for producing fumaric acid, comprising culturing Rhizopus sp. In a medium containing sorbitol.
＜２＞ソルビトールを含有する培地でリゾプス属菌を培養することを含む、リゾプス属菌のフマル酸生産能の向上方法。 <2> A method for improving the fumaric acid-producing ability of Rhizopus sp., Comprising culturing Rhizopus sp. In a medium containing sorbitol.
＜３＞ソルビトールを含有する培地でリゾプス属菌を培養することを含む、リゾプス属菌のエタノール生産能の低減方法。 <3> A method for reducing ethanol production ability of Rhizopus sp., Comprising culturing Rhizopus sp. In a medium containing sorbitol.
＜４＞上記培地に含まれるソルビトールの量が、該培地に含まれる炭素源の全量中、好ましくは１０質量％以上、より好ましくは１０～１００質量％、さらに好ましくは５０～１００質量％である、＜１＞～＜３＞のいずれか１項記載の方法。 <4> The amount of sorbitol contained in the medium is preferably 10% by mass or more, more preferably 10 to 100% by mass, and further preferably 50 to 100% by mass in the total amount of the carbon source contained in the medium. <1> to <3>.
＜５＞上記培地における上記炭素源の量が、
　好ましくは１％（ｗ／ｖ）以上、より好ましくは５％（ｗ／ｖ）以上、さらに好ましくは７．５％（ｗ／ｖ）以上であって、かつ好ましくは４０％（ｗ／ｖ）以下、より好ましくは３０％（ｗ／ｖ）であるか、あるいは、
　好ましくは１～４０％（ｗ／ｖ）、より好ましくは５～４０％（ｗ／ｖ）、さらに好ましくは７．５～４０％（ｗ／ｖ）、さらに好ましくは１～３０％（ｗ／ｖ）、さらに好ましくは５～３０％（ｗ／ｖ）、なお好ましくは７．５～３０％（ｗ／ｖ）である、
＜４＞記載の方法。 <5> The amount of the carbon source in the medium is
Preferably it is 1% (w / v) or more, more preferably 5% (w / v) or more, more preferably 7.5% (w / v) or more, and preferably 40% (w / v). Or less, more preferably 30% (w / v), or
Preferably 1 to 40% (w / v), more preferably 5 to 40% (w / v), still more preferably 7.5 to 40% (w / v), still more preferably 1 to 30% (w / v) v), more preferably 5-30% (w / v), still more preferably 7.5-30% (w / v),
<4> The method described.
＜６＞好ましくは、上記炭素源が糖類であり、かつ上記ソルビトールの量が、該糖類の全量中、好ましくは１０質量％以上、より好ましくは１０～１００質量％、さらに好ましくは５０～１００質量％である、＜４＞又は＜５＞記載の方法。 <6> Preferably, the carbon source is a saccharide, and the amount of the sorbitol is preferably 10% by mass or more, more preferably 10 to 100% by mass, and still more preferably 50 to 100% by mass in the total amount of the saccharide. % <4> or <5>.
＜７＞好ましくは、上記糖類が糖及び／又は糖アルコールである、＜６＞記載の方法。 <7> The method according to <6>, wherein the saccharide is preferably a sugar and / or a sugar alcohol.
＜８＞上記糖類がソルビトールを含有し、かつ
　ソルビトール以外の糖類を含む場合、好ましくはグルコース、マルトース、でんぷん加水分解物、フルクトース、キシロース、キシリトール及びスクロースからなる群より選択される少なくとも１種をさらに含有し、より好ましくは、グルコース、キシリトールもしくはフルクトースを含有し、さらに好ましくはグルコースを含有する、＜７＞記載の方法。 <8> When the saccharide contains sorbitol and contains a saccharide other than sorbitol, preferably at least one selected from the group consisting of glucose, maltose, starch hydrolysate, fructose, xylose, xylitol and sucrose <7> The method according to <7>, further comprising glucose, xylitol or fructose, more preferably glucose.
＜９＞上記培地における上記ソルビトールの含有量が、該培地全量中、好ましくは１～４０％（ｗ／ｖ）、より好ましくは５～３０％（ｗ／ｖ）である、＜１＞～＜８＞のいずれか１項記載の方法。 <9> The content of the sorbitol in the medium is preferably 1 to 40% (w / v), more preferably 5 to 30% (w / v) in the total amount of the medium. <1> to < 8. The method according to any one of 8>.
＜１０＞好ましくは、上記リゾプス属菌が、Ｒｈｉｚｏｐｕｓ　ｄｅｌｅｍａｒ、Ｒｈｉｚｏｐｕｓ　ｏｒｙｚａｅ、Ｒｈｉｚｏｐｕｓ　ａｒｒｈｉｚｕｓ、Ｒｈｉｚｏｐｕｓ　ｃｈｉｎｅｎｓｉｓ、Ｒｈｉｚｏｐｕｓ　ｎｉｇｒｉｃａｎｓ、Ｒｈｉｚｏｐｕｓ　ｔｏｎｋｉｎｅｎｓｉｓ、及びＲｈｉｚｏｐｕｓ　ｔｒｉｔｉｃｉからなる群より選択されるいずれか１種以上である、＜１＞～＜９＞のいずれか１項記載の方法。 <10> Preferably, the Rhizopus genus is selected from the group Rhizopus delemar, Rhizopus oryzae, Rhizopus arrizus, Rhizopus chinensis, Rhizopus nigricans, Rhizopisti. The method according to any one of> to <9>.
＜１１＞好ましくは、上記リゾプス属菌がペレットの形態であるか、又は担体に固定化されている、＜１＞～＜１０＞のいずれか１項記載の方法。 <11> The method according to any one of <1> to <10>, wherein the Rhizopus bacterium is preferably in the form of a pellet or immobilized on a carrier.
＜１２＞好ましくは、上記培養の時間が１２～２４０時間である、＜１＞～＜１１＞のいずれか１項記載の方法。 <12> The method according to any one of <1> to <11>, wherein the culture time is preferably 12 to 240 hours.
＜１３＞好ましくは、上記培養で得られた培養物からフマル酸を回収することをさらに含む、＜１＞、及び＜４＞～＜１２＞のいずれか１項記載の方法。 <13> Preferably, the method according to any one of <1> and <4> to <12>, further comprising recovering fumaric acid from the culture obtained in the above culture.
　以下、実施例に基づき本発明をさらに詳細に説明するが、本発明はこれに限定されるものではない。 Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited thereto.
実施例１　リゾプス属菌培養によるフマル酸の製造
（１）リゾプス属菌の調製
　リゾプス属菌（Ｒｈｉｚｏｐｕｓ　ｄｅｌｅｍａｒ　ＪＣＭ５５５７）のペレットを調製した。国際公開公報第２０１３／１５７３９８号の製造例１～製造例３を参考に、胞子懸濁液からペレットを調製した。 Example 1 Production of fumaric acid by culturing Rhizopus (1) Preparation of Rhizopus genus Rhizopus delmar JCM5557 pellets were prepared. Pellets were prepared from the spore suspension with reference to Production Examples 1 to 3 of International Publication No. 2013/157398.
（２）リゾプス属菌の培養
　（１）で得られたリゾプス属菌の湿菌体６．０ｇを、２００ｍＬ容三角フラスコに供したフマル酸生産評価用無機培養液４０ｍＬ（組成：１０．０％炭素源、０．１％硫酸アンモニウム、０．０６％リン酸２水素カリウム、０．０２５％硫酸マグネシウム・７水和物、０．００９％硫酸亜鉛・７水和物、５．０％炭酸カルシウム、いずれも濃度は％（ｗ／ｖ））に接種し、３５℃、１７０ｒｐｍにて撹拌培養した。培養液の炭素源としては、表１に示すグルコース、ソルビトール、又はそれらの混合物を用いた。菌体接種直後（０．５時間）及び１２０時間後に、菌体を含まない培養液上清を回収し、後述する参考例１に記載の手順にてフマル酸、リンゴ酸、コハク酸、及びエタノールの定量を行った。結果を表１に示す。グルコースのみを炭素源とする培地に比べて、ソルビトールを添加したリゾプス属菌培養物ではフマル酸生産性が向上しており、またエタノールの生産量が低下する傾向にあった。 (2) Cultivation of Rhizopus spp. Inorganic culture solution for evaluation of fumaric acid production in which 6.0 g of Rhizopus spp. Wet cells obtained in (1) were subjected to a 200 mL Erlenmeyer flask (composition: 10.0%) Carbon source, 0.1% ammonium sulfate, 0.06% potassium dihydrogen phosphate, 0.025% magnesium sulfate heptahydrate, 0.009% zinc sulfate heptahydrate, 5.0% calcium carbonate, In any case, the concentration was inoculated to% (w / v)) and cultured with stirring at 35 ° C. and 170 rpm. As the carbon source of the culture solution, glucose, sorbitol, or a mixture thereof shown in Table 1 was used. Immediately after inoculation (0.5 hours) and 120 hours later, the culture supernatant containing no bacterial cells was collected, and fumaric acid, malic acid, succinic acid, and ethanol were collected by the procedure described in Reference Example 1 described later. Was quantified. The results are shown in Table 1. Compared to a medium using only glucose as a carbon source, the Rhizopus culture added with sorbitol had improved fumaric acid productivity and decreased ethanol production.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
参考例１　有機酸及びエタノールの定量
　培養上清中の有機酸（フマル酸、リンゴ酸、コハク酸）、及びエタノールの定量は、ＨＰＬＣにより行った。
　ＨＰＬＣ分析に供する培養液上清試料は、予め３７ｍＭ硫酸にて適宜希釈した後、ＤＩＳＭＩＣ－１３ｃｐ（０．２０μｍセルロースアセテート膜、ＡＤＶＡＮＴＥＣ製）又はアクロプレップ９６フィルタープレート（０．２μｍＧＨＰ膜、日本ポール製）を用いて不溶物の除去を行なった。
　ＨＰＬＣの装置は、ＬａＣｈｒｏｍ　Ｅｌｉｔｅ（日立ハイテクノロジーズ製）を用いた。分析カラムには、ＩＣＳｅｐ　ＩＣＥ－ＩＯＮ－３００　Ｇｕａｒｄ　Ｃｏｌｕｍｎ　Ｃａｒｔｒｉｄｅ（４．０ｍｍ　Ｉ．Ｄ．×２．０ｃｍ、ＴＲＡＮＳＧＥＮＯＭＩＣ製）を接続した有機酸分析用ポリマーカラムＩＣＳｅｐ　ＩＣＥ－ＩＯＮ－３００（７．８ｍｍＩ．Ｄ．×３０ｃｍ、ＴＲＡＮＳＧＥＮＯＭＣ製）を用い、溶離液は１０ｍＭ硫酸、流速０．５ｍＬ／分、カラム温度５０℃の条件にて溶出を行なった。各有機酸、及びエタノールの検出は、それぞれ示唆屈折率検出器（ＲＩ検出器）及びＵＶ検出器（検出波長２１０ｎｍ）を用いた。濃度検量線は、標準試料〔フマル酸（販売元コード０６３－００６５５、和光純薬工業製）、リンゴ酸（販売元コード１３５－００５６２、和光純薬工業製）、コハク酸（販売元コード１９４－０４３３５、和光純薬工業製）、及びエタノール（製品番号０５７－００４５６、和光純薬工業製）〕を用いて作成した。それぞれの濃度検量線に基づいて、各成分の定量を行なった。 Reference Example 1 Quantification of organic acid and ethanol The organic acid (fumaric acid, malic acid, succinic acid) and ethanol in the culture supernatant were quantified by HPLC.
A culture supernatant sample to be subjected to HPLC analysis is appropriately diluted with 37 mM sulfuric acid in advance, and then DISMIC-13cp (0.20 μm cellulose acetate membrane, ADVANTEC) or Acroprep 96 filter plate (0.2 μm GHP membrane, Nippon Pole) ) Was used to remove insoluble matter.
As a HPLC apparatus, LaChrom Elite (manufactured by Hitachi High-Technologies) was used. ICSep ICE-ION-300 Guard Column Cartridge (4.0 mm ID × 2.0 cm, manufactured by TRANSGENOMIC) connected to the analytical column is a polymer column for organic acid analysis ICSep ICE-ION-300 (7.8 mm I.D.). D. × 30 cm, manufactured by TRANSGENOMC) was used, and the eluent was eluted under conditions of 10 mM sulfuric acid, a flow rate of 0.5 mL / min, and a column temperature of 50 ° C. Each organic acid and ethanol were detected using a suggested refractive index detector (RI detector) and a UV detector (detection wavelength 210 nm), respectively. Concentration calibration curves consist of standard samples [fumaric acid (sales code 063-00655, manufactured by Wako Pure Chemical Industries), malic acid (sales code 135-00562, manufactured by Wako Pure Chemical Industries), succinic acid (sales code 194- 04335, manufactured by Wako Pure Chemical Industries, Ltd.) and ethanol (product number 057-00456, manufactured by Wako Pure Chemical Industries, Ltd.)]. Based on each concentration calibration curve, each component was quantified.

Claims (10)

  1. 　ソルビトールを含有する培地でリゾプス属菌を培養することを含む、フマル酸の製造方法。 A method for producing fumaric acid, comprising culturing Rhizopus sp. In a medium containing sorbitol.
  2. 　前記培地に含まれるソルビトールの量が、該培地に含まれる炭素源の全量中１０質量％以上である、請求項１記載の方法。 The method according to claim 1, wherein the amount of sorbitol contained in the medium is 10% by mass or more based on the total amount of carbon sources contained in the medium.
  3. 　前記炭素源が糖類である、請求項２記載の方法。 The method according to claim 2, wherein the carbon source is a saccharide.
  4. 　前記培地中の前記糖類の量が１～４０％（ｗ／ｖ）である、請求項３記載の方法。 The method according to claim 3, wherein the amount of the saccharide in the medium is 1 to 40% (w / v).
  5. 　前記培地中のソルビトールの含有量が１～４０％（ｗ／ｖ）である、請求項１～４のいずれか１項記載の方法。 The method according to any one of claims 1 to 4, wherein the content of sorbitol in the medium is 1 to 40% (w / v).
  6. 　前記リゾプス属菌がＲｈｉｚｏｐｕｓ　ｄｅｌｅｍａｒである請求項１～５のいずれか１項記載の方法。 The method according to any one of claims 1 to 5, wherein the Rhizopus genus is Rhizopus delemar.
  7. 　前記培養の時間が１２～２４０時間である、請求項１～６のいずれか１項記載の方法。 The method according to any one of claims 1 to 6, wherein the culture time is 12 to 240 hours.
  8. 　前記培養で得られた培養物からフマル酸を回収することをさらに含む、請求項１～７のいずれか１項記載の方法。 The method according to any one of claims 1 to 7, further comprising recovering fumaric acid from the culture obtained in the culture.
  9. 　ソルビトールを含有する培地でリゾプス属菌を培養することを含む、リゾプス属菌のフマル酸生産能の向上方法。 A method for improving the fumaric acid-producing ability of Rhizopus sp., Comprising culturing Rhizopus sp. In a medium containing sorbitol.
  10. 　ソルビトールを含有する培地でリゾプス属菌を培養することを含む、リゾプス属菌のエタノール生産能の低減方法。 A method for reducing the ability of Rhizopus to produce ethanol, comprising culturing Rhizopus sp. On a medium containing sorbitol.
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