WO2023063337A1 - 実質上純粋なｄ－タリトールまたはアリトールの製造方法 - Google Patents

実質上純粋なｄ－タリトールまたはアリトールの製造方法 Download PDF

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WO2023063337A1
WO2023063337A1 PCT/JP2022/037982 JP2022037982W WO2023063337A1 WO 2023063337 A1 WO2023063337 A1 WO 2023063337A1 JP 2022037982 W JP2022037982 W JP 2022037982W WO 2023063337 A1 WO2023063337 A1 WO 2023063337A1
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allitol
talitol
allulose
microorganism
burkholderia
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French (fr)
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志郎 加藤
健 何森
明秀 吉原
進 望月
和也 秋光
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Kagawa University NUC
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    • C—CHEMISTRY; METALLURGY
    • 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; 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
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C—CHEMISTRY; METALLURGY
    • 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
    • C12N9/90—Isomerases (5.)
    • 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
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric

Definitions

  • the present invention relates to a method for producing substantially pure D-talitol or allitol from a mixture of D-talitol and allitol obtained by hydrogenating D-allulose.
  • D-allulose is the D-form of allulose, which is classified as ketohexose.
  • D-allulose also known as D-psicose, is an epimer of D-fructose and is similar in sweetness to D-fructose, but its sweetness is about 70% that of sugar, giving it a refreshing and sharp sweetness.
  • D-allulose has been proven to be zero kcal (Non-Patent Document 3) in human studies (Non-Patent Document 6) and animal studies (Non-Patent Document 7), respectively.
  • Non-Patent Document 4 Postprandial blood sugar suppressing effect
  • D-glucose which constitutes digestible sugar ingested with food and beverages
  • Patent Document 5 anti-obesity effect
  • D-allulose is available by any means, including those extracted from nature, those synthesized by chemical or biological methods, and the like.
  • D-allulose is a rare sugar and was very difficult to obtain in large quantities, but now it is possible to produce high-purity D-allulose in large quantities through a reaction using epimerase. rice field.
  • D-allulose can be produced more efficiently than D-fructose using D-allulose 3-epimerase (DAE), and can be produced in large quantities with a high performance liquid chromatography (HPLC) purity of almost 100%. It is possible to produce it (Non-Patent Document 1).
  • Ketose 3-epimerase is an enzyme that catalyzes the isomerization (epimerization reaction) of the hydroxyl group at the 3-position of ketohexose.
  • EC 5.1.3.30 is an enzyme that epimerizes D-allulose to D-fructose and D-fructose to D-allulose.
  • DAEs from multiple microorganisms have been found with equilibrium reaction ratios of D-allulose: D-fructose ranging from 27:73 to 33:67.
  • An object of the present invention is to produce pure D-talitol from a mixture of sugar alcohols D-talitol and allitol obtained by hydrogenating D-allulose.
  • D-talitol is intended to be efficiently mass-produced in a pure form as a raw material for D-tagatose, and allitol, which is a by-product of the D-talitol production stage, can also be a raw material for D-allulose.
  • allulose has an anti-obesity effect equal to or greater than that of D-allulose.
  • this separation of allitol by crystallization is a process of reducing the content of allitol.
  • the mixed solution with an increased D-talitol content and a decreased allitol content compared to before separation is passed through a chromatography column to collect a D-talitol fraction containing no allitol. or by treating with a microorganism that acts on allitol but not on D-talitol to degrade allitol to obtain substantially pure D-talitol.
  • the mixture (A) of D-talitol and allitol is reacted with a microorganism capable of producing D-allulose from allitol to produce D-allulose from allitol, which is passed through a chromatography column. , a D-talitol fraction free of D-allulose can be collected. D-talitol and D-allulose can be purified relatively easily because their chromatographic elution positions are farther apart than those of D-talitol and allitol.
  • the gist of the present invention is the method for producing substantially pure D-talitol (B) or allitol (C) of the following (1) to (8).
  • a method for producing substantially pure D-talitol (B) or allitol (C) from a mixture (A) of D-talitol and allitol comprising: (a) Crystals (A-1) with a higher allitol ratio than before crystallization and crystals (A-1) with a higher ratio of allitol than before crystallization, and obtaining a mixed solution (A-2) in which the content of D-talitol is increased and the content of allitol is decreased;
  • the mixed solution (A-2), i) collect an allitol-free D-talitol fraction through a chromatography column, or ii) treat with an allitol-affecting but not D-talitol-affecting microorganism to degrade allitol.
  • the mixture (A) of D-talitol and allitol is prepared by using D-allulose as a raw material and reducing D-allulose with hydrogen under a metal catalyst under high temperature and pressure to obtain a mixture of D-talitol and allitol.
  • the method according to (1) above which is obtained by a hydrogenation step to produce (3)
  • the crystal (A-1) having a higher allitol ratio than before the crystallization is subjected to a repeated crystallization operation, or the crystal having a higher allitol ratio is washed with an allitol solution to obtain a substantially pure crystal.
  • a microorganism (a) ii) that acts on allitol but does not act on D-talitol a microorganism (a) that has the ability to produce D-allulose from allitol and at the same time has DAE is selected.
  • D-allulose converted from allitol by action is converted to D-fructose by the action of DAE and is metabolically decomposed in the bacteria to degrade allitol, or has the ability to produce D-allulose from allitol.
  • a microorganism (b) that does not have DAE is selected, immobilized DAE is allowed to coexist, and D-allulose converted from allitol by the action of allitol is converted to D-fructose outside the cells by the action of DAE.
  • the genus Burkholderia, the genus Enterobacter, the genus Agrobacterium The method according to any one of (1) to (4) above, wherein the microorganism is selected from the group consisting of bacteria belonging to the genus Agrobacterium, the genus Buttiauxella, the genus Lelliottia, and the genus Pantoea. . (6) the microorganism is Burkholderia lata, Burkholderia multivorans, Burkholderia diffusa, Enterobacter hormaechei, Enterobacter solibacter ), Agrobacterium pusense, Buttiauxella brennerae, Butiauxella sp. (5) above, which is selected from the group consisting of the bacterial species Buttiauxella sp., Lelliottia jeotgali, and Pantoea sp.
  • the microorganism having the ability to produce D-allulose from allitol in (a) above and having DAE at the same time is Burkholderia lata, Burkholderia diffusa, Agrobacterium psensus (Agrobacterium pusense), the method according to any one of (4) to (6) above.
  • the microorganism having the ability to produce D-allulose from allitol in (b) and having no DAE is Burkholderia lata, Burkholderia multivorans, Burkholderia multivorans Burkholderia diffusa, Enterobacter hormaechei, Enterobacter soli, Buttiauxella brennerae, Buttiauxella sp. (Buttiauxella sp.), Lelliottia jeotgali, and Pantoea sp.
  • pure D-talitol which is a raw material for D-tagatose, which is one of the rare sugars, and allitol, a by-product of the production of D-talitol, can be easily mass-produced.
  • the by-product allitol can be converted to the starting material D-allulose and reused. Hydrogenation is adopted for mass production using D-allulose as a raw material, so D-allulose becomes allitol and D-talitol, and a mixture of D-talitol and allitol is the starting material.
  • This allitol is treated with a microorganism capable of producing D-allulose from allitol but not capable of converting D-talitol to convert allitol to D-allulose, which is passed through a chromatography column to obtain D-allulose.
  • - Mass production of D-talitol becomes possible by collecting the D-talitol fraction containing no allulose.
  • allitol is a by-product of the D-talitol production stage, but it can also be used as a raw material for D-allulose. There is a technical contribution to efficient mass production in a variety of forms.
  • FIG. 1 is a diagram illustrating the allitol-degrading process of microorganisms that have the ability to convert to DAE at the same time with the Izumofleet formula.
  • an allitol-degrading step using a microorganism that degrades allitol but cannot degrade D-talitol to obtain only pure D-talitol, wherein the microorganism (b) converts allitol to D-allulose It is a microorganism that has the ability to convert to DAE and does not have DAE, and is a drawing explaining the allitol degradation process in the coexistence of immobilized DAE with the Izumofleet formula.
  • Non-Patent Documents 1 and 2 The basic rules of the Izumofleet formula shown in Non-Patent Documents 1 and 2 are shown. That is, for aldoses, ketoses, and polyols, the Fischer projection formula and the Izmo-fleet structure are compared to show the notation principle of the Izmo-fleet structure, which is a pattern graphic. For example, D-glucose displayed in the Fisher projection formula, when divided into combinations of elements other than carbon to which each carbon is bonded, "CHO”, “HC-OH”, “HO-C- H”, “HC-OH”, “HC-OH” and "CH 2 OH". Details of each corresponding pattern diagram are shown in the lower part of FIG.
  • FIG. 4 shows that the pattern corresponding to each combination with the element other than carbon to which the carbon is bonded is determined in advance, and the combination of each carbon contained in the sugar and the element other than carbon to which the carbon is bonded. , and patterns.
  • the combination of aldehyde groups “CHO” corresponds to a pattern of circles with short vertical bars at the bottom.
  • a combination of ketone groups “C ⁇ O” corresponds to a pattern of circles with short vertical bars above and below.
  • D-talitol which is the raw material for D-tagatose, one of the rare sugars, and allitol, which is a by-product of the D-talitol production stage, are produced in pure form.
  • the mixture of D-talitol and allitol is prepared from D-allulose as a raw material, and D-allulose is subjected to high temperature and high pressure treatment under a metal catalyst.
  • D-allulose has a relationship with a mixture of D-talitol and allitol and raw material D-allulose, and when allitol is removed from the reaction system, a microorganism having the ability to produce D-allulose from allitol is allowed to act. , in relation to the product of allitol in the production of D-allulose.
  • D-allulose contaminating as a by-product of D-tagatose when D-allulose is produced by allowing microorganisms capable of producing D-allulose from allitol to act on allitol, a byproduct of the D-talitol production stage. , obtained as a mixture of D-tagatose and D-allulose.
  • D-allulose can be separated to obtain substantially pure D-tagatose, which can be used as a mixture for food applications.
  • Allitol and D-talitol are polyols (sugar alcohols) and rare sugars. D-talitol is oxidatively converted to D-tagatose by the action of acetic acid bacteria. Therefore, in the production of D-tagatose, D-talitol is a raw material, but allitol is a by-product and is removed from the reaction system. Allitol is a sugar alcohol with 6 carbon atoms produced by reduction of D-allulose, and it is thought that it exists dynamically back and forth through oxidation-reduction with D-allulose in zuina, a deciduous shrub.
  • the present invention adopts the process of hydrogenation for mass production, takes D-allulose as raw material, and obtains a mixture of allitol and D-talitol.
  • raw material D-allulose is reduced with hydrogen under high temperature and high pressure in the presence of a metal catalyst to produce a mixture of D-talitol and allitol (see Patent Document 3).
  • the method of industrially reducing (hydrogenating) the raw material D-allulose at low cost was adopted.
  • the metal catalyst used is a catalyst containing a metal selected from the elements of groups 8 to 10 of the periodic table.
  • Elements of Groups 8 to 10 of the Periodic Table refer to elements of the iron, cobalt, nickel and platinum groups.
  • the platinum group elements refer to the six elements of ruthenium, rhodium, palladium, osmium, iridium, and platinum.
  • metals selected from elements of Groups 8 to 10 of the periodic table metals selected from nickel and platinum group elements are preferably used as catalysts in the present invention.
  • metals selected from nickel, ruthenium, platinum and palladium are preferable in terms of hydrogenation ability. It was confirmed that ruthenium and platinum have a higher hydrogenation ability than palladium, that these reactions can be hydrogenated under low temperature and low pressure conditions, and that they have higher catalytic ability than copper chromium, Raney cobalt, and the like.
  • a so-called Raney nickel catalyst obtained by treating an alloy of nickel and aluminum or the like with caustic alkali or the like is preferable because it increases the reaction rate by increasing the surface area, that is, its catalytic activity is high.
  • the properties of the catalyst used for the reduction reaction are not only determined by the type of metal, but are also affected by the carrier on which the metal is carried.
  • supports for supporting the metal catalyst used include activated carbon, metal oxides such as titanium oxide and alumina, barium sulfate, and diatomaceous earth.
  • the mode of implementation of this step is not particularly limited, but usually, the raw material D-allulose is dissolved in a solvent such as water, and the above-mentioned metal catalyst is added to the solution.
  • the hydrogenation reaction of D-allulose is carried out by pressurizing.
  • Water is usually used as the solvent, but alcohol solvents such as ethanol, methyl acetate, ethyl acetate, mixed solvents thereof, and mixed solvents of these and water can also be used.
  • the concentration of D-allulose in the reaction solution is usually 1 to 60 w/v%, preferably 5 to 50 w/v%.
  • the reaction temperature is usually 10 to 150°C, preferably 10 to 70°C, more preferably 30 to 60°C, so as to make it difficult to produce reaction by-products.
  • the reaction pressure is generally 1 to 200 kg/cm 2 (gauge pressure), preferably 5 to 100 kg/cm 2 .
  • the progress of the reaction in this step can be confirmed by sampling the reaction solution at regular intervals and analyzing D-allulose, the sugar alcohol D-talitol and allitol to be produced.
  • HPLC or the like is preferably used for the analysis of D-allulose and the sugar alcohol D-talitol and allitol to be produced.
  • the catalyst can be easily removed by filtration, decantation, centrifugation, filtration and the like. By removing the catalyst in this manner, a solution containing the desired sugar alcohol is obtained.
  • an optically active substance for inducing diastereoisomerism that is, an asymmetric source
  • the production ratio of sugar alcohols can be changed.
  • chiral sources include boric acid, cinchonidine, cinchonine, ephedrine, quinidine, brucine, etc., alkaloids such as quinine and strychnine, sugars such as D-mannitol and derivatives thereof, menthol, camphor, terpene, and L-tartaric acid.
  • hydroxy acids such as L-lactic acid and L-malic acid
  • amino acids such as L-leucine, L-cystine and L-cysteine.
  • a synthetic asymmetric source molecularly designed for the purpose of diastereoisomeric reduction together with a catalyst containing a metal selected from nickel, ruthenium, platinum and palladium.
  • Such synthetic asymmetric sources include existing asymmetric phosphines such as BINAP.
  • the production ratio of D-talitol and allitol can be changed depending on the type of metal catalyst used. That is, the production ratio of two or more sugar alcohols changes depending on conditions such as the type of catalyst metal and the type of support.
  • D-talitol and allitol when Raney nickel is used, D-talitol:allitol is preferably produced in a ratio of about 50:50 to 40:60, and when platinum is used, D-talitol is preferred. : Allitol is preferable for obtaining a production ratio of about 30:70 to 42:58. In this way, a mixture rich in D-talitol can be easily obtained, making it easier to obtain pure D-talitol if necessary.
  • allitol crystallizes out when the mixture is concentrated, leaving the remaining mixture with less concentrated allitol relative to D-talitol.
  • this separation of allitol by crystallization is a process of reducing the content of allitol.
  • the reaction solution was sampled at regular intervals and subjected to HPLC analysis to obtain the results of obtaining the ratios of allitol and D-talitol in the water of crystallization and the mother liquor.
  • the crystallization operation By repeating the crystallization operation from , it is possible to preferentially crystallize allitol, and it is also possible to obtain pure allitol.
  • High-purity allitol can also be obtained by washing primary crystals obtained from a mixed aqueous solution of allitol and D-talitol with an allitol solution. By repeating this crystallization operation, crystals with a higher proportion of allitol than before crystallization are obtained, and finally 100% allitol crystals are obtained.
  • the obtained allitol can also be used as raw material D-allulose, and in recent years it has been reported to have an anti-obesity effect equal to or greater than that of D-allulose, so it can be used as a functional substance. .
  • the mixed solution with an increased D-talitol content and a decreased allitol content compared to before crystallization was i) passed through a chromatography column to obtain D-talitol containing no allitol.
  • Substantially pure D-talitol can be obtained by collecting the fraction or ii) degrading allitol by treatment with a microorganism that acts on allitol but not on D-talitol. In i) above, it becomes easier to purify and separate D-talitol and allitol from the remaining mixture of allitol and D-talitol by chromatography.
  • a microorganism that decomposes allitol but does not act on D-talitol is used.
  • a microorganism capable of producing D-allulose from allitol and at the same time having DAE is selected.
  • D-allulose converted from allitol within the microorganism is further converted to D-fructose by DAE and metabolically degraded within the microorganism.
  • microorganism (b) As the microorganism (b), a microorganism (b) having the ability to produce D-allulose from allitol and not having DAE is selected. As shown in FIG. 3, when the microorganism of (b) is allowed to act on allitol in the presence of immobilized DAE, D-allulose converted from allitol inside the microorganism is transformed into D- by the action of DAE outside the microorganism. It is converted to fructose, enters the cells and is metabolically decomposed, and allitol is decomposed.
  • Step of producing D-allulose from allitol by allowing a microorganism capable of producing D-allulose from allitol Further, after the hydrogenation step, as (a), a microorganism capable of producing D-allulose from allitol is allowed to act on the aqueous solution of the mixture (A) of D-talitol and allitol to convert D-allulose from allitol. can be manufactured.
  • Microorganisms having the ability to produce D-allulose from allitol include Burkholderia, Enterobacter, Agrobacterium, Butiaucthera, which do not have the ability to convert D-talitol.
  • the microorganism is Burkholderia ratta lata) ⁇ ⁇ (Burkholderia multivorans) ⁇ ⁇ (Burkholderia diffusa) ⁇ ⁇ (Enterobacter hormaechei) ⁇ ⁇ (Enterobacter soli) ⁇ ⁇ (Agrobacterium pusense) ⁇ ⁇ (Buttiauxella brennerae), Buttiauxella sp. (Buttiauxella sp.), Lelliottia jeotgali, and Pantoea sp. (Pantoea sp.).
  • D-talitol and D-allulose exist outside the cells. This is passed through a chromatography column to collect a D-talitol fraction containing no D-allulose. D-Talitol and D-allulose can be purified relatively easily because their chromatographic elution positions are separated.
  • Microorganisms that have the ability to produce D-allulose from allitol and do not have a DAE include Burkholderia spp., Enterobacter spp., A microorganism selected from the group consisting of bacteria belonging to the genera Buttiauxella, Lelliottia, and Pantoea.
  • Microorganisms having the ability to produce D-allulose from allitol and at the same time having a DAE belong to the genus Burkholderia or Agrobacterium, which do not have the ability to convert D-talitol as described above. Examples include Burkholderia lata, Burkholderia diffusa, and Agrobacterium pusense.
  • the present inventors used a soil library to search for microorganisms capable of producing D-allulose from allitol upon contact with an aqueous solution containing allitol.
  • any bacterium belonging to the genus Burkholderia that has been confirmed to have the ability to produce D-allulose from allitol can be used as long as it has the ability.
  • Burkholderia martivorans S332-4 1 strain
  • Burkholderia martivorans Y555-2d 2 strains
  • Burkholderia diffusa Y452-1 3 strains dated April 30, 2021, respectively.
  • strains were subsequently deposited on February 1, 2022 under accession numbers NITE BP-03413, NITE BP-03410, NITE BP-03412, NITE BP-03408, NITE BP-03414, NITE BP-03411.
  • Bacteria of the genus Enterobacter having the ability to produce D-allulose from allitol can be used as long as they are of the genus Enterobacter and have that ability.
  • Bacterial species of the genus Enterobacter having that ability include, for example, Enterobacter hormaechei, Enterobacter soli, and the like.
  • Enterobacter formaekei BCr11-1, Enterobacter formaekei BCr11-2, and Enterobacter sori BDr27-1 which have been confirmed to have the ability to produce D-allulose from allitol, were acquired from Kisarazu, Chiba Prefecture on April 30, 2021.
  • bacteria belonging to the genus Agrobacterium, the genus Buttiauxella, the genus Lelliottia, and the genus Pantoea having the ability to produce D-allulose from allitol are the genus Agrobacterium, the genus Butiauxella , Reliotia, and Pantoea can be used.
  • Bacterial species of the genera Agrobacterium, Butiauxella, and Reliotia that have that ability include, for example, Agrobacterium pusense, Buttiauxella brennerae, Butiauxella sp. (Buttiauxella sp.), Lelliottia jeotgali, and Pantoea sp.
  • Butiauchera brenellae BCr16-1-3 strain, Butiauxera sp. BCr16-1-1 strain, Reliotia geogari NH309-4 strain, Reliotia geogari Ou92-1-1 strain, and Reliotia geogari T33-1 strain were each acquired on April 30, 2021 at 2-Kazusa Kamatari, Kisarazu City, Chiba Prefecture.
  • these bacteria are first cultured in a normal nutrient medium, preferably under aerobic conditions such as shaking, aeration and agitation, and allowed to grow. Allitol in the mixture is converted to D-allulose during the culture or using the obtained viable cells. Bacteria used for the oxidation of D-allulose can utilize it in the culture medium. In addition, live cells separated from the culture solution and dried cells can also be used. As a culture method, a nutrient medium, preferably a liquid medium, containing a nutrient source required by these bacteria, such as a carbon source, a nitrogen source, an inorganic salt, yeast extract, etc., is added. Bacteria are inoculated and cultured under aerobic conditions at a temperature of 20-40° C. for 1-10 days.
  • a nutrient medium preferably a liquid medium, containing a nutrient source required by these bacteria, such as a carbon source, a nitrogen source, an inorganic salt, yeast extract, etc.
  • the viable cells obtained by such a culture method are brought into contact with an aqueous solution containing allitol, preferably under predetermined conditions such as shaking, aeration and stirring, and injecting oxygen to convert allitol to D-allulose.
  • an aqueous solution containing allitol preferably under predetermined conditions such as shaking, aeration and stirring, and injecting oxygen to convert allitol to D-allulose.
  • these bacteria can be used after being immobilized, and highly active immobilized bacteria can be obtained by various immobilization methods such as carrier binding, cross-linking, gel entrapment, and microencapsulation. can be done.
  • HPLC analysis was performed using a GL-C611 column (manufactured by Hitachi Ltd.) and Prominence (manufactured by Shimadzu Corporation) under the conditions of an eluent of 0.1 mM-NaOH, a temperature of 60°C and a flow rate of 1 mL/min.
  • a device manufactured by Shimadzu Corporation, trade name “RID-20A” was used.
  • OD 600 which is an index of cell concentration, was calculated by measuring absorbance at a wavelength of 600 nm using an ultraviolet-visible spectrophotometer UV-1800 (manufactured by Hitachi, Ltd.).
  • Example 1 100 g of a 20% NaOH aqueous solution was added to 10 g of 50% Raney nickel (manufactured by Wako Pure Chemical Industries, Ltd.). After the addition, the mixture was heated at 90°C for 1 hour. After confirming that the generation of bubbles had stopped, the catalyst was washed with distilled water by decantation. Washing was performed until the washing liquid reached pH 9.2.
  • 300 g of an aqueous solution containing 100 g of D-allulose was added with 24 g of Raney nickel obtained by the above method. was adjusted to 600 g. At that time, calcium carbonate was added to adjust the pH of the reaction solution to 7.
  • the temperature was kept at 50° C., the hydrogen pressure was kept at 12 kg/cm 2 (gauge pressure), and the stirring speed was kept at 700 rpm to carry out the reaction.
  • the reaction solution was sampled at regular time intervals and analyzed for D-allulose, D-talitol and allitol to confirm the progress of the reaction. Analysis of D-allulose, D-talitol and allitol was performed using HPLC. Samples subjected to analysis were obtained by appropriately diluting the reaction solution sampled over time, and desalted by adding an anion exchange resin and a cation exchange resin. As a result, D-allulose decreased to 1% after 8 hours of reaction, and D-talitol and allitol were obtained at a production ratio of 55:45.
  • Example 2 2050 g of a mixed aqueous solution of allitol and D-talitol obtained by reducing 270 g of D-allulose to 100% by hydrogenation using Raney nickel as a catalyst was concentrated and crystallized, and separated from the mother liquor by suction filtration under reduced pressure to recover crystals. Let this be the primary crystal. The primary crystals were recrystallized and the same procedure was repeated to obtain secondary and tertiary crystals. 48 g of allitol was obtained by three crystallization operations. The tertiary crystal obtained was found to have an allitol purity of 100% by the same analytical method as that shown in Example 1.
  • DAE D-allulose 3-epimerase
  • Recombinant E. coli cells expressing DAE derived from strain Y586-1 were suspended in 10 ml of 50 mM Tris-HCl buffer (pH 7.5), and the cell suspension was cooled in ice water with an ultrasonic homogenizer. After crushing, the crushed product was centrifuged at 12000 rpm for 30 minutes, and the centrifugal supernatant was used as a crude enzyme solution.
  • the crude enzyme solution was heat-treated at 60° C. for 10 minutes to inactivate contaminating proteins and centrifuged to remove them.
  • the partially purified enzyme after the heat treatment was added to a weakly basic anion exchange resin A111S (manufactured by Purolite) ion exchange resin (immobilizing carrier) previously equilibrated with 50 mM Tris-HCl buffer (pH 7.5) to immobilize the enzyme. turned into The resulting immobilized DAE was used for subsequent tests.
  • Example 4 A 1% polyol mixture was reacted with Reliotia geogari, a microorganism capable of producing D-allulose from allitol.
  • the cells were washed with sodium phosphate buffer (10 mM, pH 7.0).
  • reaction solution was sampled over time, removed by centrifugation, desalted with an ion-exchange resin, and then analyzed for sugar composition using HPLC. The results are shown in FIG. After 18 hours of reaction, the allitol peak disappeared (retention time 19.2 min), and a highly pure D-talitol (retention time 21.8 min) solution was obtained.
  • Example 6 Reaction using 20% polyol mixture
  • BDr27-3-2 strain cells cultured in TSB medium were collected by centrifugation and washed with sodium phosphate buffer (10 mM, pH 7.0).

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WO2002092545A1 (en) * 2001-05-11 2002-11-21 Fushimi Pharmaceutical Company, Limited Method for producing sugar alcohol
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JP2009207462A (ja) * 2008-03-06 2009-09-17 Unitika Ltd 合成原料用糖の製造方法
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