JP5030045B2 - Method for producing disaccharide containing rare sugar - Google Patents

Description

  The present invention relates to a novel disaccharide containing a rare sugar as a constituent monosaccharide and a synthesis method thereof.

  Sucrose phosphorylase is an enzyme that acts on sucrose in the presence of inorganic phosphate to produce α-glucose-1-phosphate and D-fructose. Conversely, sucrose can be synthesized from α-glucose-1-phosphate and D-fructose. It has also been reported that glucosyl-L-sorbose can be synthesized from α-glucose-1-phosphate and L-sorbose by sucrose phosphorylase (Non-patent Document 1).

  Various physiological activities of rare sugars are being revealed by mass production (Patent Documents 2 and 3, Non-Patent Documents 2 and 3). For example, D-psicose has attracted attention as a sugar that does not promote fat synthesis and does not accumulate body fat, particularly intraperitoneal fat, compared to simple sugars such as D-glucose and D-fructose (Non-Patent Document 2). . It has also been reported that the effective energy value of D-psicose is almost zero. As described above, various physiological activities of rare sugars are being revealed by mass production.

  However, there have been no reports of synthesis of sucrose phosphorylase with α-glucose-1-phosphate and D-psicose, D-tagatose, D-sorbose, L-tagatose, L-psicose, or L-fructose. . In the first place, it seems that these sugars were not included in the study because they were difficult to obtain.

Japanese Patent Laid-Open No. 2002-345458 JP 2004-269359 A Japanese Patent Laid-Open No. 2004-300079 Doudoroff, M. The Enzymes, 2nd. Ed. (Boyer, P.D., Lardy, H., Myrbaeck, K., eds.) Pp.229-236 1961 Matsuo T, et al., Asia Pacific J. Clin. Nutr. 10, 233-237, 2001 Matsuo T, et al., J. Nutr. Sci. Vitaminol 48, 77-80, 2002

  An object of the present invention is to provide a novel disaccharide containing a rare sugar as a constituent monosaccharide and a synthesis method thereof. A novel disaccharide (oligosaccharide) containing a rare sugar whose various physiological activities are becoming clear can be expected to have a new physiological activity.

  Sucrose is a disaccharide in which D-glucose and D-fructose are combined. If it is possible to synthesize disaccharides having different constituent sugars, a new application is expected. However, that method is not an easy reaction. The reason is that the structure of D-glucose or D-fructose cannot be changed with the disaccharide bound. Therefore, the most probable method is to combine two monosaccharides that constitute the disaccharide with some reaction. Moreover, in order to couple | bond two monosaccharides, since the energy of coupling | bonding is required, it is necessary to give it to a reaction system with a certain method. Thus, these problems could be solved by using a reverse reaction of sucrose phosphorylase. That is, it is possible to convert sucrose D-fructose into rare ketose by using D-glucose-1-phosphate as the energy supply and various rare ketoses as new constituent sugars.

The gist of the present invention is a method for producing a disaccharide containing a rare sugar as a constituent monosaccharide of the following (1) to ( 3 ).
(1) Sucrose phosphorylase is allowed to act on a ketohexose selected from α-glucose-1-phosphate and a rare sugar to synthesize a disaccharide containing a rare sugar as a constituent monosaccharide, and the target disaccharide is obtained from the reaction mixture. In a method for producing a disaccharide containing a rare sugar as a constituent monosaccharide,
Ketohexose selected from the above rare sugars is D-psicose,
The above-mentioned reaction in which sucrose phosphorylase is allowed to act on α-glucose-1-phosphate and D-psicose is a reaction in which sucrose phosphorylase and D-tagatose 3 epimerase are allowed to act using sucrose as a starting material in the presence of inorganic phosphate,
A disaccharide containing a rare sugar as a constituent monosaccharide is α-D-glucopyranosyl- (1 → 2) -β-D-psicofuranoside having a molecular weight of 342.30 and having the following structure and the following specific light intensity . A method for producing disaccharides.
[Specific light intensity]
Specific light intensity glucosyl-D-psicose at 20 ° C. and wavelength of 589 nm using a photometer P-1030ST manufactured by JASCO Corporation: [α] ₂₀ = 74.36
Sucrose as a control: [α] ₂₀ = 66.78
( 2 ) The method for producing a disaccharide according to (1) above, wherein the sucrose phosphorylase is derived from a bacterium belonging to Leuconostoc mesenteroides .
( 3 ) The method for producing a disaccharide according to ( 2 ) above, wherein the sucrose phosphorylase is immobilized on a carrier.

The enzyme used in the present invention is sucrose phosphorylase. Sucrose phosphorylase is an enzyme that synthesizes sucrose from glucose-1-phosphate and D-fructose and binds with Glucose 1 → 2 Fructose. D-glucose and D-fructose are each independently in four forms in an aqueous solution. When sucrose is formed, glucose is bound in the form of α-glucopyranoside and fructose in the state of β-fructofuranoside. D-psicose also takes four forms, but it is presumed that the same binding mode is obtained when D-psicose is used instead of D-fructose.

Sucrose phosphorylase is a known enzyme, and any enzyme that produces α-glucose 1-phosphate and fructose from sucrose and inorganic phosphate and / or a salt thereof can be used. Sucrose phosphorylase is naturally contained in various microorganisms. Examples of microorganisms producing sucrose phosphorylase, Leuconostoc mesenteroides, Streptococcus thermophilus, Streptococcus mutans, Streptococcus pneumoniae, Pseudomonas sp., Clostridium sp., Pullularia pullulans, Acetobacter xylinum, Agrobacteium sp., Synecococcus sp., E. Coli, Aspergillus Examples include , but are not limited to, niger , Monilia sitophila , Sclerotinea escerotiorum, and Chlamydomonas sp.

  In this specification, the phrase “derived from” a microorganism does not mean that the enzyme is isolated directly from the microorganism, but that the enzyme is obtained by utilizing the microorganism in some form. Say. For example, when an enzyme gene of the microorganism is introduced into E. coli and the enzyme is isolated from the E. coli, the enzyme is said to be “derived” from the microorganism.

In the present invention, in addition to using commercially available products, those obtained by culturing microorganisms that produce these enzymes can also be used. Specifically, sucrose phosphorylase produced by microorganisms such as Leuconostoc mesenteroides and Pseudomonas saccharophila can be used. The former sucrose phosphorylase is an enzyme classified in EC 2.4.1.7 according to the report of the International Biochemical Union Enzyme Committee, and sucrose is phosphorolyzed in the presence of phosphoric acid to produce glucose-1- It is a catalyst for a reversible reaction that produces phosphoric acid and fructose.

In addition, all the enzymes used in this specification are enzymes derived from Leuconostoc mesenteroides .
The sucrose phosphorylase used in the method of the present invention can be prepared, for example, as follows. First, a microorganism that produces sucrose phosphorylase is cultured. This microorganism may be a microorganism that directly produces sucrose phosphorylase. Alternatively, a gene encoding sucrose phosphorylase may be cloned, a microorganism advantageous for sucrose phosphorylase expression may be genetically recombined with the obtained gene to obtain a recombinant microorganism, and sucrose phosphorylase may be obtained from the obtained microorganism. . As sucrose phosphorylase, a purified enzyme or an unpurified enzyme may be used as it is, but a known immobilization means such as a carrier binding method, a crosslinking method, a gel inclusion method, a microencapsulation method, etc. may be used as an immobilized enzyme. Good. In addition, live cells can be immobilized by a global immobilization method using polyacrylamide, κ-carrageenan, alginic acid, a photocrosslinkable resin prepolymer, or the like, and used as a biocatalyst.

  The commercially available sucrose phosphorylase used in the present invention has a good specific activity, and in a preferred embodiment, the specific activity is 70 units / mg or more. In the present specification, an enzyme other than sucrose phosphorylase, a solvent, an additive, and the like may be included as long as the state of sucrose phosphorylase is not lost.

As used herein, “activity (U)” refers to an enzyme activity that converts 1 micromolar substrate per minute. Since the activity is usually indicated by U for commercially available enzymes, the amount of enzyme used in this specification is indicated based on that.
The amount of sucrose phosphorylase used is not particularly limited, and the optimum amount of enzyme used is determined in consideration of economy, but the concentration of sucrose phosphorylase usually contained in the solution is typically per reaction volume. It is 0.1U-10U, More preferably, it is 0.5U-8U. If the amount of sucrose phosphorylase is too large or too small, a sufficient amount of disaccharide may not be obtained.

D-psicose is still expensive due to the emergence of epimerase in recent years (for example, see Japanese Patent Application Laid-Open No. 6-125576), such as being able to be produced from D-fructose, which is a monosaccharide abundant in nature. However, it became relatively easy to obtain. It has been found that by using D-psicose as a raw material, the problem of raw material cost can be solved, and a hetero-oligosaccharide that can be expected to have a new physiological activity based on D-fructose can be obtained.
Among monosaccharides, D-psicose is a hexasaccharide having a ketone group as a reducing group. This D-psicose is known to have D-form and L-form as optical isomers. Here, D-psicose is a known substance but rarely exists in nature. Therefore, it is defined as “rare sugar” according to the definition of the International Society of Rare Sugars.

Describing rare sugars, rare sugars can be defined as monosaccharides (aldoses, ketoses and sugar alcohols) that rarely exist in nature. Also in the present invention, it is a rare sugar based on the above definition, preferably ketose D-psicose.
This definition is ambiguous because it is not defined by the structure or properties of sugar. That is, there is no definition of an amount such as a rare sugar that is a certain amount or less. However, in general, there are six types of aldoses that exist in large quantities in nature: D-glucose, D-galactose, D-mannose, D-ribose, D-xylose, and L-arabinose. Other aldoses are defined as rare sugars. The As ketose, D-fructose exists, and other ketoses can be said to be rare sugars. Other ketoses include D-tagatose, D-sorbose, L-fructose, L-psicose, L-tagatose, and L-sorbose.
Sugar alcohols can be produced by reducing monosaccharides, but D-sorbitol is relatively large in nature, but the others are quantitatively small, so these are also rare sugars.

  The rare saccharide used as a raw material in the present invention has been difficult to obtain per se, but a method for producing a rare saccharide from a monosaccharide existing in large quantities in the natural world is being developed. can do.

  In the enzyme reaction of the present invention, glucose-1-phosphate is present in the reaction system. The concentration of glucose-1-phosphate in the reaction solution is not particularly limited, but is 0.001 mM or more and 1 M or less, preferably 0.01 mM or more and 500 mM or less, more preferably 0.1 mM or more and 200 mM or less.

<Reaction substrate>
The α-glucose-1-phosphate and the rare sugar ketohexose used in the method of the present invention are preferably pure.
The concentration of α-glucose-1-phosphate contained in the reaction solution is typically 1 to 20%, more preferably 5 to 10%. The concentration in this specification is calculated by Weight / Volume, that is, (weight of α-glucose-1-phosphate (g)) × 100 / (volume of solution (ml)).
The concentration of ketohexose which is various rare sugars contained in the reaction solution is typically 1 to 20%, more preferably 5 to 10%. The concentration in this specification is calculated by Weight / Volume, that is, (weight of ketohexose (rare sugar) (g)) × 100 / (volume of solution (ml)).

As shown in Example 2 described later, since glucose-1-phosphate is expensive, the possibility of synthesizing glucosyl-D-psicose by a conjugation reaction with sucrose phosphorylase and D-tagatose 3 epimerase using sucrose as a starting material investigated.
D-tagatose 3-epimerase is a D-ketohexose 3-epimerase having the activity of epimerizing the 3-position of D-ketohexose to produce the corresponding D-ketohexose, and has the following physicochemical properties (special (See Kaihei 6-125776).
(1) Action and substrate specificity The 3-position of D-ketohexose is epimerized to produce the corresponding D-ketohexose. Epimerize position 3 of D- or L-ketopentose to produce the corresponding D- or L-ketopentose.
(2) Optimal pH and pH stability It has an optimal pH at pH 7 to 10, and is stable at pH 5 to 10.
(3) Optimal temperature and thermal stability Optimal temperature near 60 ° C, stable at 50 ° C or less.
(4) An absorption band is shown in the ultraviolet absorption spectrum 275 to 280 nm.
Bacteria having the ability to produce D-ketohexose 3-epimerase belonging to the genus Pseudomonas such as Pseudomonas chicory ST-24 (FERM BP-2736) and its mutants are cultured in a nutrient medium and D-ketohexose 3-epimerase. Is produced and collected.

<Solvent>
The solvent that dissolves the sucrose phosphorylase and the carbohydrate that is the substrate may be any solvent as long as it does not impair the enzyme activity of sucrose phosphorylase. A typical solvent is water. The solvent is preferably an aqueous solution such as a pH buffer solution such as Tris-HCl buffer solution, phosphate buffer solution, 3- (N-monophorino) propane sulfonic acid (MOPS) buffer solution in order to maintain enzyme activity. The pH range is typically 4 to 11, more preferably 6 to 8.

  Although the reaction time is not particularly limited, it may be terminated when the yield of the desired hetero-oligosaccharide is maximized, and the concentration of sucrose as a raw material and the enzyme concentration are usually in the range of 1 minute to several hundred hours. May be appropriately determined in consideration of the above. After completion of the enzyme reaction, the heterooligosaccharide can be separated by a known method as necessary. In the method described above, since the enzyme is contained in the reaction solution, usually, the reaction solution is first heated to deactivate the enzyme, and then the heterooligosaccharide is separated by an appropriate separation means. At this time, if sucrose or the like remaining in the reaction solution hinders separation, it is decomposed in advance using an appropriate decomposing enzyme. For example, unreacted sucrose is decomposed by adding invertase to the reaction solution. Thereafter, the heterooligosaccharide can be purified by applying purification means such as gel filtration chromatography and activated carbon column chromatography, if desired.

  In the present invention, a specific heterooligosaccharide is produced corresponding to the rare sugar used as a raw material. For example, when D-psicose is used as the rare sugar, α-D-glucose- (1 → 2) β-D-psicose (glucosyl-D-psicose) is produced.

A disaccharide containing a rare sugar as a constituent monosaccharide obtained by the synthesis method of the present invention will be described.
The difference in structure between sucrose and glucosyl-D-psicose is that the directions of H and OH are reversed at the 2-position of carbon (indicated by a circle).
1. Structure General name: Sucrose
Official name: α-D-glucopyranosyl- (1 → 2) -β-D-fructofuranoside
(Α-D-glucopyranosyl- (1 → 2) -β-D-fructofuranoside)


Generic name: Glucosyl-D-psicose Glucosyl-D-psicose Official name: α-D-glucopyranosyl- (1 → 2) -β-D-psicofuranoside
(Α-D-Glucopyranosyl- (1 → 2) -β-D-psicofuranoside)

The carbon numbers of sucrose shown in Chemical Formula 5 are as shown in Chemical Formula 7 below, with D-glucose on the left and D-fructose on the right. Sucrose has no reducing property because the 1-position of D-glucose and the 2-position of D-fructose are bonded. α-glucose is reducible because the carbon at position 1 must be an aldehyde group in the ring-opened structure, and β-fructose is reducible in the ring-opened structure. It is necessary that the carbon keto group changes to a hydroxyl group, the hydroxyl group of the 1st carbon becomes a keto group, and the 1st carbon becomes an aldehyde group. However, in the sucrose molecule, the 1-position carbon of α-glucose and the 2-position carbon of β-fructose, which are necessary to show reducibility, are glycosidically bonded with an oxygen atom in between. Absent. Therefore, it does not show reducibility.

2. Properties The chemical formulas for glucosyl-D-psicose and sucrose are both C ₁₂ H ₂₂ O ₁₁ and the molecular weight is 342.30. From the fact that sucrose does not show reducing power by the Somogy Nelson method (0.0153 (g / w) 0.0153), it is clear that glucosyl-D-psicose shows almost no reducing power (0.0485). Glucosyl-D-psicose is strongly presumed to have a glycosidic bond at the 1-position of α-glucopyranoside and 2-position of β-psicofuranoside, similar to sucrose.

  Hereinafter, the present invention will be described in more detail with reference to examples. The present invention is not limited to these examples.

1) Enzyme reaction conditions A total of 8 kinds of the reaction compositions shown in Table 1 were synthesized using commercially available sucrose phosphorylase (manufactured by Oriental Yeast Co., Ltd.). One of the 8 species used D-fructose as a control (synthesizes sucrose when synthesized). The synthesis efficiency with the other seven species was compared.

  Table 2 shows the ratio and retention time of disaccharides produced from each substrate by high performance liquid chromatography (HPLC) analysis of the resulting disaccharides.

A Hitachi HPLC column GL-C611 (manufactured by Hitachi Chemical Co., Ltd.) was used as the column, and a differential refractometer (L-7490 refractive index detector, manufactured by Hitachi High-Tech) was used as the detector. The column was kept at 60 ° C., and 10 ⁻⁴ M sodium hydroxide solution was used as an eluent at a flow rate of 1.0 mL / min. The obtained signals were collected using data analysis software (SmartChrom, manufactured by KYA Technologies) and analyzed using the software.

  According to the results obtained by the applicants so far, in the above-mentioned apparatus and conditions, almost all kinds of monosaccharides such as glucose and psicose are eluted in about 30 minutes after 15 minutes, and sucrose and lactose And other disaccharides elute from 13 to 15 minutes. There is no HPLC data for the synthetic disaccharides described in this specification except for ketohexose other than D-fructose (in this case, sucrose), but as shown in Table 2. It can be considered that disaccharides are produced from the retention time.

  The resulting disaccharides are glucosyl-D-fructose (sucrose) from D-fructose, glucosyl-D-psicose, glucosyl-D-tagatose, glucosyl-D-sorbose, glucosyl-L- Fructose, glucosyl-L-psicose, glucosyl-L-tagatose, glucosyl-L-sorbose. However, the minor peaks seen in D-tagatose, L-tagatose and L-psicose are likely to be disaccharides with slightly different binding modes. Furthermore, the peak at 17.7 minutes seen when L-tagatose is used corresponds to the position where the monosaccharide is usually eluted, but so far it has not been isolated and analyzed for its structure.

  FIG. 1 shows the results of HPLC analysis of disaccharides produced when D-fructose or D-psicose is used as a substrate.

2) Separation conditions Of the eight disaccharides obtained in Table 2, glucosyl-D-psicose synthesized with D-psicose was purified and further characterized. Since sucrose phosphorylase catalyzes an equilibrium reaction, glucosyl-D-psicose, which is a product, and α-glucose-1-phosphate, D-glucose, and D-psicose, which are products, are present in the solution after the reaction. include. In order to purify glucosyl-D-psicose, it is first necessary to deactivate the enzyme by heating the reaction solution after the enzyme reaction. The heating time is typically 1 minute to 10 minutes, more preferably 3 minutes to 5 minutes. The heating may be performed by any means, but it is preferable to perform the heating while stirring so that the heat is uniformly transmitted to the entire solution. The solution is stirred, for example, in a beaker. A water bath is mentioned as an apparatus used for a heating.

The obtained glucosyl-D-psicose was separated and purified using Dowex 50W-X2 (Dowex) resin. This separation and separation resin has the same separation and elution pattern as the above-mentioned HPLC column GL-C611. A reaction solution having a sugar concentration of 10% and 2 ml was separated on a column having a diameter of 0.8 cm × 120 cm at a flow rate of 0.5 ml / min. The amount of sample that can be separated is 1 to 3 ml at a sugar concentration of 5% to 20%, more preferably 2 ml at 10%. The fraction collector was set at 1.5 ml / tube and 120 fractions were collected. FIG. 2 shows a HPLC chart of the purified glucosyl-D-psicose. In HPLC, glucosyl-D-psicose seems to have undergone some thermal decomposition because the column is heated at 60 degrees. The purity in this result is 95% (Table 3: Purity of purified glucosyl-D-psicose).

(Synthesis of glucosyl-D-psicose by conjugation with sucrose phosphorylase and D-tagatose 3 epimerase)
Since glucose-1-phosphate is expensive, the possibility of synthesizing glucosyl-D-psicose by sucrose phosphorylase and D-tagatose 3-epimerase in the presence of inorganic phosphate was studied. This principle is sulrose phosphorylase, and sucrose is decomposed into D-glucose 1-phosphate and D-fructose. The D-fructose is converted to D-psicose by D-tagatose 3-epimerase. The reaction proceeds using D-glucose 1-phosphate and D-psicose produced in this reaction as substrates. This eliminates the need to add D-glucose 1-phosphate as a substrate. Table 4 shows the reaction composition of each enzyme, substrate and buffer. Inorganic phosphoric acid is introduced into the system by additives such as enzyme preparations.

[result]
The result of HPLC is shown in FIG. 14.40 minutes was glucosyl-D-psicose, and it was successfully synthesized about 10%.

(Properties of glucosyl-D-psicose)
The properties of the purified glucosyl-D-psicose were investigated.
1. Specific light intensity The specific light intensity of glucosyl-D-psicose and sucrose as a control was measured. For the measurement, a photometer P-1030ST manufactured by JASCO Corporation is used.
The specific light intensity at a temperature of 590 nm and a wavelength of 589 nm was measured.
Glucosyl-D-psicose: [α] ₂₀ = 74.36
Sucrose: [α] ₂₀ = 66.78

2. Quantification of reducing power The amount of reducing sugars in sucrose as glucosyl-D-psicose and as a control was quantified by the Somogene Nelson method. In addition, it measured at 0.01% (g / w).
Glucosyl-D-psicose: 0.0485
Sucrose: 0.0153
Note) At this time, glucosyl-D-psicose contained some 0.3% of D-glucose on HPLC, so some reducing sugar was detected, but glucosyl-D-psicose itself has no reducing power. To be judged. When the value 0.0485 of glucosyl-D-psicose is simply calculated from the reducing power of D-glucose, about 0.0002% glucose is present. Together, these results are that glucosyl-D-psicose has no reducing power. That is, the binding mode indicates that the 1-position of D-glucose and the 2-position of D-psicose are bound.

3. ¹³ C-NMR
The binding mode of sucrose is α1 glucose (pyranose ring) -β2 fructose (furanose ring). It is presumed that glucosyl-D-psicose has a similar binding mode because it is bound by sucrose phosphorylase. From the NMR results (FIG. 4) and the measurement results of reducing power, it is presumed that glucosyl-D-psicose is bound by α1 glucose (pyranose ring) -β2 psicose (furanose ring).

4). Effect on yeast invertase Invertase (β-fructofuranosidase) is an enzyme that breaks down sucrose, the same binding mode as sucrose, and one of the constituent sugars, D-fructose, was converted to D-psicose. With structure. Compared to sucrose, the only difference is the position of the 3-position OH of D-fructose. Therefore, glucosyl-D-psicose is highly likely to be recognized as a substrate-like substance by invertase. So glucosyl-D-psicose is yeast
Whether it was degraded by invertase from Saccharomyces cerevisiae , the inhibitory activity was examined conversely. The enzyme reaction conditions are shown in Table 5.
<Supplementary explanation of Table 5>
10 + 30 μl of each sugar + water means that 30 μl of water is added to 10 μl of each sugar, 20 + 20 μl means that 20 μl of water is added to 20 μl of each sugar, and 40 + 0 μl means that only 40 μl of each sugar is added. 10 + 30 μl has a final concentration of 2.5 mM, 20 + 20 μl has a final concentration of 5 mM, and 40 + 0 μl has a final concentration of 10 mM.
Immediately after the reaction, the invertase reaction was stopped by heat treatment in the same manner as described above, and the composition of each saccharide was quantified by HPLC as before, and the enzyme activity was evaluated by the amount of D-fructose produced. .

[result]
The results are shown in Table 6.
It was revealed that invertase was inhibited by glucosyl-D-psicose in a concentration-dependent manner.

(Prospects for commercialization)
Glucosyl-D-psicose is a novel disaccharide that has not been synthesized and elucidated. What is expected from being not degraded by invertase is expected to be degraded and digested by higher organisms and difficult to be absorbed. This can be expected to have a wide range of uses as a new biomaterial, including new sweeteners.
As the results of specific studies show, application as an invertase inhibitor can be expected. That is, since glucosyl-D-psicose is a substance in which the D-fructose portion of sucrose is replaced with D-psicose, the inhibitory activity of invertase, which is a sugar-degrading enzyme, is higher than that of D-psicose. Since this inhibitory activity inhibits the decomposition of sucrose in the intestinal tract, it is expected that the amount of energy will be reduced by not digesting and absorbing sucrose.

In addition, in order to mass-produce, a large amount of glucose-1-phosphate and D-psicose as raw materials are required. D-psicose has been successfully mass-produced, but glucose-1-phosphate is expensive, so its acquisition is key. As an example of a method for obtaining D-glucose 1-phosphate, when cellobiose phosphorylase is allowed to act on cellobiose, which is a degradation product of cellulose, glucose-1-phosphate can also be used from cellobiose.
In addition, it can be obtained at low cost by using phosphorylases of various glucosyl compounds, and is expected to be achieved by establishing optimum conditions for constructing a system coupled with this reaction.

Results of HPLC analysis of disaccharides produced when D-fructose or D-psicose is used as a substrate. (A) For D-fructose (B) For D-psicose HPLC chart of glucosyl-D-psicose after purification. The HPLC analysis result after reaction which performed the conjugation reaction of D-tagatose 3-epimerase and sucrose phosphorylase using sucrose as a substrate. Glucosylpsicose ¹³ C-NMR results.

Claims (3)

Sucrose phosphorylase is allowed to act on ketohexose selected from α-glucose-1-phosphate and a rare sugar to synthesize a disaccharide containing a rare sugar as a constituent monosaccharide, and the target disaccharide is separated from the reaction mixture. In a method for producing a disaccharide containing a rare sugar as a constituent monosaccharide,
Ketohexose selected from the above rare sugars is D-psicose,
The above-mentioned reaction in which sucrose phosphorylase is allowed to act on α-glucose-1-phosphate and D-psicose is a reaction in which sucrose phosphorylase and D-tagatose 3 epimerase are allowed to act using sucrose as a starting material in the presence of inorganic phosphate,
A disaccharide containing a rare sugar as a constituent monosaccharide is α-D-glucopyranosyl- (1 → 2) -β-D-psicofuranoside having a molecular weight of 342.30 and having the following structure and the following specific light intensity . A method for producing disaccharides.
[Specific light intensity]
Specific light intensity glucosyl-D-psicose at 20 ° C. and wavelength of 589 nm using a photometer P-1030ST manufactured by JASCO Corporation: [α] ₂₀ = 74.36
Sucrose as a control: [α] ₂₀ = 66.78 The sucrose phosphorylase is Leuconostoc mesenteroides ( Leuconostoc
is derived from bacteria belonging to mesenteroides), method for producing a disaccharide according to claim 1. The method for producing a disaccharide according to claim 2 , wherein the sucrose phosphorylase is immobilized on a carrier.