JP5075376B2 - Process for producing 1,5-D-anhydroglucitol - Google Patents

Description

  The present invention relates to a method for producing 1,5-D-anhydroglucitol by a microorganism.

1,5-D-anhydroglucitol (hereinafter 1,5-AG) is a polyol having a structure in which the hydroxyl group at the 1-position of glucose is reduced, and since many animals and plants biosynthesize it, It is a widely distributed substance. There is a report that 1,5-AG is metabolically stable in the animal body and the amount excreted in exhaled breath for 48 hours is 1% or less of the total (see Non-patent Document 1). Can be used as a sweetener. Furthermore, it is used industrially as a research reagent and a clinical test reagent.
As a method for preparing 1,5-AG, a chemical synthesis method from β-D-glucopyranose pentaacetate has been reported (see Non-Patent Document 2). The synthesis method is carried out by dissolving β-D-glucopyranose pentaacetate in ether, followed by Br conversion with hydrogen bromide and deacetylation with lithium aluminum hydride.

Also reported is a method for preparing 1,5-AG by extracting, isolating, and crystallizing 1.5-AG from protea leaves with organic solvents such as ethanol and hexane. (See Non-Patent Document 3).
These chemical synthesis and extraction from plants are multi-stage and complicated processes, and organic solvents such as ether and hexane are used. The need arises. Furthermore, questions remain from the viewpoint of safety.
As a means for solving these problems, a production method is required which has a simple production process and does not use an organic solvent such as ether.

1,5-AG has been reported to be biosynthesized by Escherichia coli (see Non-Patent Document 1). However, biosynthesis by Escherichia coli is not intended for industrial production, and the amount of 1,5-AG produced is about μg / L, which is not satisfactory for industrial production.
As another method for preparing 1,5-AG, hydrogenation to 1,5-AF in the presence of a palladium catalyst has been reported (see Non-Patent Document 4). A reaction product is generated, and 1,5-AG is about 1/5 of the reaction product and is difficult to obtain efficiently.
1,5-D-anhydrofructose (hereinafter 1,5-AF) is a saccharide that can be prepared by degrading α-1,4-glucan such as starch with α-1,4-glucan lyase, In recent years, a technology capable of mass production at low cost has been proposed (see Patent Document 1). Using this 1,5-AF as a starting material, a production process was simple and a production method using no organic solvent such as ether was examined.
JP 2005-168454 A Biochemistry Vol. 69, No. 12, pp. 1361-1372, (1997) J. et al. Am. Chem. Soc. 72, 4547-4553 (1950) Phytochemistry Vol. 22, No. 9, 1959-1960 (1983) Carbohydrate Research 337 (2002) 873-890

  The present invention has been made from the above viewpoint, and an object of the present invention is to use a microorganism having the ability to produce 1,5-AG from 1,5-AF using 1,5-AF as a starting material. It is to provide a method of manufacturing.

  Other objects and advantages of the present invention will become apparent from the following description.

As a result of intensive studies, the present inventors have found a microorganism having an ability to convert 1,5-AF to 1,5-AG, and produces 1,5-AG by a simple process and without using an organic solvent. The present invention has been reached.
That is, the present invention contacts 1,5-AF with a microorganism having the ability to convert 1,5-AG to convert 1,5-AF to 1,5-AG, and the resulting 1,5-AG is This is a method for producing 1,5-AG, characterized by sampling.

  According to the present invention, 1,5-anhydroglucitol can be industrially advantageously produced from 1,5-AF by a microorganism culture method without using an organic solvent.

In the present invention, the microorganism having the ability to convert 1, 5-AF to 1, 5-AG, Sa Kkaromaisesu genus Saccharomyces Mycobacterium death genus, Willi Opsys genus, Brettanomyces genus, Candida genus, Garakutomaisesu genus, Pitia, Tigosaccharomyces, Schizosaccharomyces, Tiretiopsis, Sterimatomyces, Kurtsumanomyces, Leukospodium, Erythrobasidium, Philobasidium, Trichosporon, Microorganisms belonging to the genus Cocovaera, Ferromyces, Breromyces, Criprococcus, Tsuchiyaja or Aureobasidium are used .

  More specifically, Saccharomyces cerevisiae, Saccharomycodes rudizi, Williopsis platensis, Brettanomyces braxylenesis, Candida valida, Candida kefir, Galactomyces chitri-auranty, Pitia anomala, Tigo saccharomyces・ Luxi, Schizosaccharomyces pombe, Tiretiopsis wasintonensis, Sterimatomyces halophyllus, Kurzmanomyces tie lay, Leukospodium scotti, Erythrobasidium Hasegawa nuran, Filovasidi Umm Floriforme, Trichosporon Cutaneum, Cocovaela Tyrandica, Ferromyces Polybolus, Breromyses Arbus, Criprococcus a Bidasu, include microorganisms such as Tsuchiyaeya Wing feel di or Aureobasidium microspheres Thich Tam.

  Hereinafter, the present invention will be described in detail. Examples of the microorganism used in the present invention include, for example, the genus Saccharomyces, Saccharomycodes, Williopsis, Brettanomyces, Candida, Galactomyces, Pitia, Tigosaccharomyces, Schizosaccharomyces, Tirethiopsis , Sterimatomyces, Kurtumanoses, Leukospodium, Erythrobasidium, Philobasidium, Trichosporon, Cocovaela, Ferromyces, Brelomyces, Criprococcus, Tutyaeja and Aureobasidi Examples include microorganisms belonging to the genus and having the ability to convert 1,5-AF to 1,5-AG.

Specific examples of the microorganisms include Saccharomyces cerevisiae, Saccharomycodes rudizi, Williopsis platensis, Brettanomyces braxylenesis, Candida valida, Candida kefir, Galactomyces chitri-auranty, Pitia anomala , Chigosaccharomyces luxi, Shizosaccharomyces pombe, Tiretiopsis washintonensis, Sterimatmyces halophyllus, Kurzmanomyces tie lay, Leukospodium scotti, Erythrobasidium hasegawa nuran, Philobasidium floriforme, Trichosporon cutaneum, Cocovaela tyrandica, Ferromyces polybolus, Breromyces albus, Kryproco Examples include Cass albidas, Tsuchiyaya wingfieldi and Aureobasidium microstitutum.
Further, specific examples of the microorganism used in the present invention include the following strains.

  Saccharomyces cerevisiae NBRC 0210, Saccharomyces ludwigii NBRC 1043, Williopsis plantensis (Williopsis plantensis) Candida valida NBRC 0159, NBRC 11015, Candida kefyr NBRC 0432, Galactomyces citri-auranti N RC 10821, Pichia anomala NBRC 0707, Tigosaccharomyces rouxii NBRC 1914, Shizosaccharomyces pombe (Shizosaccharomyces , Sterigmatomyces halophilus NBRC 1844, Kurtzmanomyces nexairei NBRC 10118, Leukospodium Scotty ridium scottii) NBRC 1212, Ellis ass Sidi Umm Hasegawanuran (Erythrobasidium hasegawanurn) NBRC 1058, Philo Basi di Umm Florianopolis formestane (Filobasidium floriforme) NBRC 1603, Trichosporon Kyutaneumu (Trichosporon cutaneum) NBRC 1198, Kokovaera Thai Randy mosquito (Kockovaella thylandica) NBRC 10521, Ferromyces polyborus NBRC 10120, Bulleromyces albus NBRC 1192, Criprococcus albida (Cryptococcus albidus) NBRC 0434, is a Tsuchiyaeya Wing feel di (Tsuchiyaea wingfieldii) NBRC 10204 and Aureobasidium microspheres Thich Tam (Aureobasidium microstictum) NBRC 32070, and the like. These strains are available at the Department of Biotechnology National Institute of Technology and Evaluation, Address C / O Incorporated A8. Kazusa-kamatari 2-home, Kisarazu-shi, Chiba, 292-0818 Japan). Any microorganism can be used as the microorganism used in the present invention as long as it has the ability to convert 1,5-AF into 1,5-AG, and is not limited to the above microorganism.

  For example, the microorganism used in the present invention is mutated by subjecting the microorganism to ultraviolet irradiation, N-methyl-N-nitrosoguanidine (NTG) treatment, ethylmethanesulfonate (EMS) treatment, nitrous acid treatment, acridine treatment, etc. It may be a mutant strain or a strain such as a genetically modified strain induced by a genetic technique such as cell fusion or genetic recombination.

As a culture solution containing 1,5-AF for culturing microorganisms, a medium containing a normal carbon source, nitrogen source, inorganic ions, and, if necessary, an organic nutrient source can be used. As the carbon source, for example, carbohydrates such as glucose, alcohols such as glycerol, organic acids, and the like are appropriately used. As organic nutrient sources, for example, yeast extract, malt extract, peptone, meat extract, corn steep liquor, casein degradation product, etc. containing vitamins, amino acids and the like are used as appropriate. As inorganic ions, for example, magnesium ions, phosphate ions, calcium ions, and the like are appropriately used. A medium for collecting 1,5-AG can be prepared by adding 1,5-AF aqueous solution sterilized separately to the medium.
The culture conditions are not particularly limited. For example, the culture can be performed at a pH of 3 to 7 and a temperature of 20 to 40 ° C. under aerobic conditions, and the culture can be performed for about 2 to 7 days while maintaining an appropriate pH and temperature.

The 1,5-AG thus produced in the culture solution is separated and purified from the culture by a well-known means that is usually performed. Specifically, after removing cells and solids by centrifugation and diatomaceous earth filtration, decolorization with activated carbon, desalting with ion exchange resin, and concentration to form a syrup. Subsequently, 1,5-AG can be separated and concentrated by appropriately combining operations such as ion exchange, adsorption, and separation by gel filtration chromatography, and cooled and crystallized to obtain. Furthermore, the obtained crystals can be dissolved in water and recrystallized to obtain polyhedral white crystals. From the measurement results of this sample by high performance liquid chromatography (hereinafter HPLC), nuclear magnetic resonance spectrum (hereinafter ¹ H-NMR) and electrospray ionization mass spectrometry (hereinafter ESI-MS), this crystal was found to be 1,5-AG. Was identified.

Since the amount of 1,5-AG produced in the culture medium can be measured quickly by HPLC, the cultivation can be terminated when the amount of 1,5-AG produced reaches the maximum. Detailed conditions of the HPLC measurement are shown below.
Separation column: Shodex SP810-MCIGELCK08S connection (Showa Denko KK, Mitsubishi Chemical Co., Ltd.), mobile phase: distilled water, flow rate: 1.0 mL / min, column temperature: 40 ° C., detection: differential refractive index detector, Sample dosage: 20 μL. As a specific example, FIG. 1 shows the measurement results of a culture solution obtained by culturing Saccharomyces cerevisiae NBRC 0210 for 5 days in a medium containing commercially available standard reagents 1,5-AG and 1,5-AF.

  EXAMPLES Hereinafter, although an Example demonstrates this invention in detail, this invention is not limited to these Examples. Unless otherwise specified,% used in the following description is capacity (w / v)%.

Example 1
500 mL of a medium containing 1.0% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract and pH 6.0 was dispensed into a 1 L culture flask and sterilized by heating at 120 ° C. for 20 minutes. .

Saccharomyces cerevisiae cultured in slant medium (glucose 1.0%, peptone 0.5%, yeast extract 0.3%, malt extract 0.3%, agar powder 1.5%, pH 6.0) in the above medium. (Saccharomyces cerevisiae) One platinum loop of NBRC 0210 was collected and cultured with shaking at 25 ° C. for 3 days. This was used as a seed culture solution. 5 L of medium having the same composition as above was placed in a 10-liter mini jar fermenter, and 1,5-AF aqueous solution subjected to sterilization through a 0.45 μm pore size filter was added to a final concentration of 2.5%. To the medium containing 1,5-AF, 500 mL of the seed culture solution was added, and the culture solution containing 1,5-AG was obtained by shaking culture at 25 ° C. for 5 days with a stirring speed of 200 rpm and an aeration rate of 1 L / min. (FIG. 1). Bacteria and solids were removed from the culture by centrifugation (3,000 rpm-10 minutes) and diatomaceous earth filtration, and then activated carbon (steam charcoal) and ion exchange resin (SK-1B: SA10AP = 2: 1). Decolorization and desalting were carried out by Mitsubishi Chemical Corporation. The purified solution was concentrated, and then subjected to column chromatography using Na ⁺ type ion exchange resin (UBK530 manufactured by Mitsubishi Chemical Corporation) as a stationary phase to separate 1,5-AG. Again, after concentrating to a sugar concentration of 45% or more, the solution was cooled at 4 ° C. to precipitate crystals. The crystals were further dissolved in distilled water and recrystallized to obtain 4.5 g of polyhedral white crystals. This crystal was dissolved in milli-Q water and analyzed by HPLC. As a result, a single symmetry peak was obtained, confirming that this crystal was a single component. Further, the substance was identified by analysis with ¹ H-NMR and ESI-MS under the following conditions.

¹ H-NMR
Commercially available standard reagent 1,5-AG and the above white crystals, observation frequency: 499.8 MHz, solvent D ₂ O, concentration 10 mg / 700 μL, reference TSP-d ₄ ((CH ₃ ) ₃ CD ₂ CD ₂ COONa) , Temperature: 25 ° C., observation width: 5 KHz, data point: 64 K (Fourier transform at 128 K), pulse width: 30 °, pulse repetition time: 10 sec, number of integrations: 8 times. 5-AG and the previous white crystal showed the same spectrum (FIG. 2).

ESI-MS
The white crystals were dissolved in Milli-Q water so as to have a concentration of 50 nmol / ml, and methanol was added so that the final concentration was 30% (V / V). Thereafter, 0.1% (V / V) formic acid was added and mass analysis was performed at an ionization voltage of 850 V. As a result, a spectrum having an ion mass-to-charge ratio of 165 (m / z) was obtained (FIG. 3). This is m = 164 (mass of 1,5-AG) +1 (mass of one proton), z = 1 (number of charges). m / z = (164 + 1) / 1 = 165. From the results of the previous HPLC (FIG. 1), ¹ H-NMR (FIG. 2), and ESI-MS (FIG. 3), white crystals were 1,5-AG. Identified.

Example 2
Dispense 5 ml of glucose 1.0%, peptone 0.5%, yeast extract 0.3%, malt extract 0.3%, pH 6.0 into a test tube and heat sterilize at 120 ° C for 20 minutes. Each of the slant culture cells shown in Table 1 was ingested by 1 platinum ear, and cultured with shaking at 25 ° C. for 3 days at 100 rpm. This was used as seed culture. 45 ml of the medium having the same composition as described above was dispensed into a 300 ml shake flask, 5 ml of the above seed culture solution was added, and cultured with shaking at 100 rpm at 25 ° C. for 6 days. After completion of the culture, the cells were removed from the medium by centrifugation, and the amount of 1,5-AG produced was measured by HPLC (1,5-AF non-added: 1,5-AF (-)). Separately, one slant cultured microbial cell shown in Table 1 was ingested under the same conditions as the above seed culture, and cultured at 25 ° C. for 3 days with shaking. 45 ml of the same composition medium was dispensed into a 300 ml shake flask, and 1,5-AF aqueous solution sterilized with a 0.45 μm pore size filter was added to 2.5%. To this medium, 5 ml of the seed culture was added, and cultured at 25 ° C. for 3 to 6 days with shaking at 100 rpm. After removing the cells by centrifugation, 1,5-AG produced in the culture solution was quantified by HPLC, and the culture was terminated when the production amount reached the maximum. (1,5-AF addition: 1,5-AF (+)) The amount of 1,5-AG in the culture solution was 3.5 to 11.4 g / L in the group in which 1,5-AF was added. On the other hand, no 1,5-AG was detected in the 1,5-AF non-added group. The results are shown in Table 1. As a specific example, FIG. 4 shows the HPLC measurement results in a culture solution of Saccharomyces cerevisiae NBRC 0210 cultured for 5 days with or without 1,5-AF.

Example 3
500 ml of 1.0% glucose, 0.5% peptone, 0.3% yeast extract, 0.3% malt extract, pH 6.0 medium was dispensed into a 1 L culture flask and sterilized by heating at 120 ° C. for 20 minutes. . Strain in which reaction products other than 1,5-AG were not observed in Example 2 above: Slope culture of galactomycins citri-auranti NBRC 10821 (glucose 1.0%, peptone One platinum loop of 0.5%, yeast extract 0.3%, malt extract 0.3%, agar powder 1.5%, pH 6.0) was collected and cultured with shaking at 25 ° C. for 3 days. This was used as seed culture. 5 L of a medium having the same composition as above was placed in a 10-liter mini jar fermenter, and 1,5-AF aqueous solution sterilized with a 0.45 μm pore size filter was added to a final concentration of 2.5%. The medium containing 1,5-AF is inoculated with 500 ml of the seed culture, shaken at 200 rpm, aerated at 1 L / min, and shaken at 25 ° C. for 6 days to give reaction products other than 1,5-AG. A culture solution not containing was obtained (FIG. 5).

  According to the present invention, 1,5-AG can be industrially produced as a food by using a microorganism using 1,5-AF as a starting material by a simple operation and without using an organic solvent or the like. Is possible.

High-performance liquid chromatography (HPLC) spectrum of a culture solution obtained by shaking culture of Saccharomyces cerevisiae NBRC 0210 in a medium containing 1,5-AF for 5 days. 500 MHz ¹ H-NMR spectrum (nuclear magnetic resonance spectrum) of the standard 1,5-AG reagent and the crystalline product obtained in Example 1. ESI-MS spectrum (electrospray ionization mass spectrum) of the same crystal product as above. Saccharomyces cerevisiae NBRC for 5 days in medium containing 1,5-AF (1,5-AF (+)) and medium without 1,5-AF (1,5-AF (-)) The high performance liquid chromatography (HPLC) spectrum of the culture solution obtained by shaking culture of 0210. High-performance liquid chromatography (HPLC) spectrum of a culture solution obtained by shaking and culturing Galactomyces citri-auranti NBRC 10821 in a culture solution containing 1,5-AF for 5 days.

Explanation of symbols

1,5-AF: 1,5-D-anhydrofructose 1,5-AG: 1,5-D-anhydroglucitol HPLC: High performance liquid chromatography
¹ H-NMR: proton-nuclear magnetic resonance spectrum ESI-MS: electrospray ionization-mass spectrometry

Claims (3)

Saccharomyces, Saccharomycodes, Williopsis, Brettanomyces, Candida, Galactomyces, Pitia, Tigosaccharomyces, Shizosaccharomyces, Tiretiopsis, Sterimatomyces, Kurtumanomai A microorganism belonging to the genus Seth, Leukospodium, Erythrobasidium, Philobasidium, Trichosporon, Cocovaela, Ferromyces, Breromyces, Cryptococcus, Tutyaeja or Aureobasidium , 1, 5-D-and anhydrofructose is contacted with a microorganism having the ability to convert 1, 5-D-anhydroglucitol glucitol to produce a 1, 5-D-anhydroglucitol glucitol, and the product 1,5-D-anhydroglucito A method for producing 1,5-D-anhydroglucitol, which comprises collecting the water. The microorganism is Saccharomyces cerevisiae, Saccharomycodes rudizi, Williopsis platensis, Brettanomyces braxylenesis, Candida valida, Candida kefir, Galactomyces chitri-auranty, Pitia anomala, Tigo saccharomyces luxi -, Schizosaccharomyces pombe, Tirethiopis vaccintonensis, Sterimatomyces halophyllus, Kurtmanomyces tieley, Leukospodium scotti, Erythrobasidium Hasegawa nuran, Philobacidium philosophy Floriforme, Trichosporon Cutaneum, Cocovaela Tyrandica, Ferromyces Polybolus, Brelomyces Arbus, Criprococcus albi Scan method as described in claim 1, which is Tsuchiyaeya wing feel di or Aureobasidium microspheres Thich Tam. Saccharomyces, Saccharomycodes, Williopsis, Brettanomyces, Candida, Galactomyces, Pitia, Tigosaccharomyces, Shizosaccharomyces, Tiretiopsis, Sterimatomyces, Kurtumanomai Using microorganisms belonging to the genus Seth, Leukospodium, Erythrobasidium, Philobasidium, Trichosporon, Cocovaela, Ferromyces, Breromyces, Cryptococcus, Tutyaeja or Aureobasidium Use of 1,5-D-anhydrofructose for the production of 1,5-D-anhydroglucitol by microbial culture methods.

Images