Method for producing cellooligosaccharides

Abstract

To provide a method in which cello-oligosaccharide can be produced inexpensively at high yield.SOLUTION: In a method for producing cello-oligosaccharide according to an embodiment, sucrose phosphorylase and cello-dextrin phosphorylase are allowed to work on at least one kind of primer selected from the group consisting of glucose, cellobiose and derivative in which their anomeric positions are modified, and sucrose in the presence of phosphoric acid. In this case, phosphoric acid concentration in a reaction system is set to 3 mol / m3 or more and 120 mol / m3 or less.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for producing cellooligosaccharides using an enzyme. [Background technology]

[0002] As a method for producing cellulose oligomers, i.e., cellooligosaccharides, using an enzyme, a synthesis method utilizing the reverse reaction of the phosphorolytic enzyme cellodextrin phosphorylase (CDP) is known.

[0003] For example, Patent Document 1 describes the synthesis of cellooligosaccharides by reacting α-glucose-1-phosphate with a primer such as glucose in a mixed solvent containing water and a water-soluble organic solvent by the action of cellodextrin phosphorylase. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2019-193601 A Summary of the Invention [Problem to be solved by the invention]

[0005] However, the above synthesis method has problems in that the starting material, α-glucose-1-phosphate, is very expensive, and the yield of cellooligosaccharides is not necessarily high.

[0006] In view of the above, an object of an embodiment of the present invention is to provide a method for producing cellooligosaccharides inexpensively and in high yield. [Means for solving the problem]

[0007] The method for producing cellooligosaccharides according to an embodiment of the present invention includes a reaction step of reacting at least one primer selected from the group consisting of glucose, cellobiose, and derivatives thereof modified at the anomeric position with sucrose phosphorylase and cellodextrin phosphorylase in the presence of phosphoric acid, and the reaction system is heated to a phosphoric acid concentration of 3 mol / m 3, including the amount of α-glucose-1-phosphate contained as an intermediate product. 3 More than 120mol / m 3 The following is a method for producing cellooligosaccharides. Effect of the Invention

[0008] According to an embodiment of the present invention, α-glucose-1-phosphate is produced from sucrose by sucrose phosphorylase, and cellooligosaccharides are produced from the α-glucose-1-phosphate thus obtained and a primer by cellodextrin phosphorylase, so that cellooligosaccharides can be produced inexpensively. In addition, by specifying the phosphoric acid concentration in the reaction system, the yield of cellooligosaccharides can be increased. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The method for producing cellooligosaccharides in this embodiment involves using at least one selected from the group consisting of glucose, cellobiose, and their derivatives modified at the anomeric position as a primer, and reacting sucrose and the primer with sucrose phosphorylase (hereinafter sometimes referred to as SP) and cellodextrin phosphorylase (hereinafter sometimes referred to as CDP) in the presence of phosphoric acid.

[0010] This reaction, as represented by the following formula, combines the reaction of SP to produce α-glucose-1-phosphate (hereinafter sometimes referred to as αG1P) from sucrose and phosphate, and the reaction of CDP to produce cellooligosaccharides from αG1P and the above primer, in which cellooligosaccharides are synthesized in two stages by the action of two types of phosphorolytic enzymes, using sucrose as the glucose donor and the above primer as the glucose acceptor.

[0011] [ka]

[0012] The first reaction is a reaction to produce αG1P by sucrose phosphorolysis enzyme SP, and αG1P can be synthesized using inexpensive sucrose as the starting material. The second reaction utilizes the reverse reaction of phosphorolysis enzyme CDP, in which αG1P is sequentially polymerized as a monomer to the glucose receptor primer, resulting in cellooligosaccharides with a cellulose type II crystal structure. This two-step synthesis makes it possible to synthesize cellooligosaccharides in a single batch from inexpensive sucrose. In addition, since the phosphate generated in the reaction by CDP is consumed in the reaction by SP, i.e., phosphate circulates in the reaction system, phosphorolysis caused by the accumulation of phosphate can be suppressed, and the yield of cellooligosaccharides can be improved.

[0013] The sucrose as the glucose donor is not particularly limited and may be naturally occurring or chemically synthesized. The initial concentration of sucrose in the reaction system (usually a reaction solution; the same applies below) (i.e., the concentration at the time of charging; the same applies below) is not particularly limited and may be, for example, 10 to 1000 mol / m 3 Or 100 to 500 mol / m 3 That's fine too.

[0014] As described above, the primer, which is a glucose receptor, is at least one selected from the group consisting of glucose, cellobiose, and derivatives thereof modified at the anomeric position. The derivative has a substituent at the anomeric position of glucose or cellobiose, more specifically, the hydroxyl group bonded to the carbon atom at the anomeric position is substituted with another organic group (substituent). Examples of the substituent include an alkoxy group, an organic group containing an azide group (including those in which an azide group is directly bonded to the carbon atom at the anomeric position), and an organic group containing an amino group (including those in which an amino group is directly bonded to the carbon atom at the anomeric position). Here, the amino group may be a primary amino group, a secondary amino group, or a tertiary amino group.

[0015] An example of the glucose derivative is represented by the following general formula (1). [ka] In formula (1), R 1 -OR 2 , -R 3 -N3 or -R 4 It is preferably represented by -NH2. The wavy line in the formula indicates that the stereochemistry at the anomeric position is α, β, or a mixture of α and β.

[0016] R 2 is preferably represented by a hydrocarbon group having 1 to 12 carbon atoms. The hydrocarbon group may be an aliphatic hydrocarbon group or an aromatic hydrocarbon group, and is preferably an alkyl group, and may have a straight chain or a branched chain. The number of carbon atoms in the hydrocarbon group is preferably 1 to 5, and more preferably 1 to 3. 2 preferably represents an alkyl group having 1 to 5 carbon atoms, and more preferably represents an alkyl group having 1 to 3 carbon atoms.

[0017] R 1 -OR 2Specific examples of the above include alkyl glucosides such as methyl glucoside, ethyl glucoside, octyl glucoside, decyl glucoside, and dodecyl glucoside, and aryl glucosides such as phenyl glucoside.

[0018] R 3 is a direct bond or -OR 5 - stands for R 5 represents a divalent hydrocarbon group having 1 to 20 carbon atoms. The divalent hydrocarbon group may be a divalent aliphatic hydrocarbon group or a divalent aromatic hydrocarbon group. Examples of the divalent aliphatic hydrocarbon group include an alkanediyl group and an alkenediyl group, and the group may have a straight chain or a branched chain. Examples of the divalent aromatic hydrocarbon group include a divalent aliphatic hydrocarbon group having an aromatic ring substituent and an arenediyl group, and the aromatic ring may have a substituent such as an alkyl group added thereto. R 5 The number of carbon atoms in R is preferably 1 to 10, and more preferably 1 to 5. 5 is preferably an alkanediyl group having 1 to 10 carbon atoms, and more preferably an alkanediyl group having 1 to 5 carbon atoms.

[0019] R 1 -R 3 Specific examples of -N3 include azidodeoxyglucoside, azidoalkylglucoside (eg, azidoethylglucoside), and the like.

[0020] R 4 is a direct bond or -OR 6 - stands for R 6 represents a divalent hydrocarbon group having 1 to 20 carbon atoms. The divalent hydrocarbon group may be a divalent aliphatic hydrocarbon group or a divalent aromatic hydrocarbon group. Examples of the divalent aliphatic hydrocarbon group include an alkanediyl group and an alkenediyl group, and the group may have a straight chain or a branched chain. Examples of the divalent aromatic hydrocarbon group include a divalent aliphatic hydrocarbon group having an aromatic ring substituent and an arenediyl group, and the aromatic ring may have a substituent such as an alkyl group added thereto. R 6The number of carbon atoms in R is preferably 1 to 10, and more preferably 1 to 5. 6 is preferably an alkanediyl group having 1 to 10 carbon atoms, and more preferably an alkanediyl group having 1 to 5 carbon atoms.

[0021] R 1 -R 4 Specific examples of -NH2 include aminodeoxyglucosides and aminoalkylglucosides (eg, aminoethylglucoside).

[0022] An example of the cellobiose derivative is represented by the following general formula (2). [ka] R in Equation (2) 7 is R in formula (1). 1 The wavy lines indicate that the anomeric configuration is α, β, or a mixture of α and β.

[0023] In one embodiment, the primer is preferably at least one selected from the group consisting of glucose and its glucose derivatives modified at the anomeric position. When glucose or its derivatives are used, there is an advantage that cellooligosaccharides are more likely to precipitate (deposit) than when cellobiose is used, making purification easier.

[0024] The initial concentration of the primer in the reaction system is not particularly limited and may be, for example, 5 to 300 mol / m 3 Or 10 to 200 mol / m 3 Or 30 to 100 mol / m 3 The molar ratio of sucrose to primer in the reaction system (sucrose / primer ratio) when charged is also not particularly limited, and may be, for example, 20 / 1 to 1 / 1, 15 / 1 to 4 / 3, or 10 / 1 to 2 / 1.

[0025] Phosphate is used in the reaction of producing αG1P by SP, and αG1P and fructose are produced from sucrose due to the presence of phosphoric acid. The phosphoric acid is not particularly limited, and may be inorganic phosphoric acid, or inorganic phosphate such as sodium dihydrogen phosphate, potassium dihydrogen phosphate, disodium hydrogen phosphate, or dipotassium hydrogen phosphate, or any one or more of these may be used in combination. Phosphate may be a phosphate buffer solution.

[0026] In this embodiment, the phosphoric acid concentration in the reaction system is 3 to 120 mol / m 3 The phosphoric acid concentration is set to 3 mol / m 3 By setting the phosphoric acid concentration at 120 mol / m or more, the reaction of producing αG1P by SP can be promoted, and the yield of cellooligosaccharides can be increased. 3 By keeping the phosphoric acid concentration in the reaction system at 7 to 80 mol / m or less, phosphorolysis by CDP can be suppressed and the yield of cellooligosaccharides can be increased. 3 is preferably 8 to 70 mol / m 3 It is.

[0027] Here, the phosphate concentration in the reaction system refers to the total phosphate concentration (molar concentration) in the reaction system including the amount contained as the intermediate product α-glucose-1-phosphate, and is the sum of the molar concentration of the inorganic phosphate and / or inorganic phosphate salt and the molar concentration of αG1P. In order to achieve the above-mentioned phosphate concentration, for example, the initial concentration of the phosphoric acid charged into the reaction system as the inorganic phosphate and / or inorganic phosphate salt is set to 3 to 120 mol / m 3 , preferably 7 to 80 mol / m 3 , more preferably 8 to 70 mol / m 3 It would be better to change it to:

[0028] The origin of sucrose phosphorylase (SP) is not particularly limited, and for example, SP derived from microorganisms such as Leuconostoc mesenteroides and Streptococcus mutans may be used.

[0029] The amount of SP used in the reaction system is not particularly limited and may be, for example, 0.001 to 10 U / mL, or 0.01 to 1 U / mL. The enzyme amount of SP is 100 mol / mL at 37° C. and pH 7. 3 of sucrose and 50 mol / m 3 The amount of enzyme that releases 1 μmol of αG1P per minute from 100 phosphate is defined as 1 U.

[0030] The cellodextrin phosphorylase (CDP) may be of any origin, and may be derived from microorganisms such as Clostridium thermocellum and Cellulomonas. As an example, CDP derived from Clostridium thermocellum YM4 can be prepared in an E. coli expression system according to the method described in M. Krishnareddy et al., J. Appl. Glycosci., 2002, 49, 1-8.

[0031] The amount of CDP used in the reaction system is not particularly limited, and may be, for example, 0.01 to 10 U / mL, or 0.1 to 1 U / mL. The amount of the CDP enzyme is 50 mol / m 3 of αG1P and 50 mol / m 3 D-(+)-cellobiose and 3-morpholinopropanesulfonic acid buffer (50 mM, pH 7.5) containing CDP diluted at a specified ratio so that the conversion rate of αG1P after a reaction time of 100 minutes is 10% or less are incubated at 37°C, and the amount of phosphate produced by CDP is quantified. The amount of enzyme that releases 1 μmol of phosphate per minute is defined as 1 U.

[0032] The enzymatic reaction using the SP and CDP in this embodiment is typically carried out in a reaction solution containing water as a solvent, and more specifically, the reaction solution is prepared by mixing sucrose, primer, phosphate, SP, and CDP together with a solvent to obtain a predetermined concentration, and then incubating the reaction solution at a predetermined temperature.

[0033] The reaction temperature is preferably 25 to 45° C. By keeping the reaction temperature at 25° C. or higher, the reaction rate of the enzyme can be increased. Also, by keeping the reaction temperature at 45° C. or lower, a decrease in the yield of cellooligosaccharides can be suppressed.

[0034] The reaction time is not particularly limited, and may be, for example, 1 hour to 15 days, or 1 day to 10 days.

[0035] The pH of the reaction solution is preferably about 5.0 to about 9.0. The pH of the reaction solution may be adjusted by adding, in addition to the phosphate buffer involved in the reaction, for example, tris(hydroxymethyl)aminomethane hydrochloride buffer, citric acid, 3-morpholinopropanesulfonic acid (MOPS), hydroxyethylpiperazineethanesulfonic acid (HEPES), etc.

[0036] In one embodiment, a mixed solvent consisting of water and a water-soluble organic solvent may be used as the solvent in the enzyme reaction. That is, SP and CDP may be reacted with the primer and sucrose in a mixed solvent containing water and a water-soluble organic solvent. By using such a mixed solvent, the polymerization degree distribution of cellooligosaccharides can be narrowed. The mixed solution is obtained, for example, by mixing a buffer solution, which is an aqueous solution, with a water-soluble organic solvent.

[0037] The water-soluble organic solvent is an organic solvent having a solubility of 5 mL or more in 100 mL of water at 20° C., and examples thereof include alcohols having 1 to 4 carbon atoms, such as methanol, ethanol, 1-propanol, isopropyl alcohol, and t-butanol; ketones, such as acetone; ethers, such as tetrahydrofuran; amides, such as N,N-dimethylformamide, N,N-dimethylacetamide, and N-methylpyrrolidone; nitriles, such as acetonitrile; and sulfoxides, such as dimethyl sulfoxide. These may be used alone or in combination of two or more. As the water-soluble organic solvent, it is more preferable to use at least one selected from the group consisting of methanol (MeOH), ethanol (EtOH), N,N-dimethylformamide (DMF), and dimethyl sulfoxide (DMSO).

[0038] The buffer solution to be mixed with the water-soluble organic solvent is not particularly limited, and examples thereof include HEPES buffer, Tris-HCl buffer, MOPS buffer, acetate buffer, and phosphate buffer. A buffer solution capable of maintaining the pH of the reaction solvent at about 5 to 9 at the reaction temperature is preferably used.

[0039] The ratio of water to the water-soluble organic solvent used is not particularly limited, and the water-soluble organic solvent is preferably 5 to 50 volume %, more preferably 5 to 30 volume %, and even more preferably 8 to 20 volume %, based on 100 volume % of the mixed solvent.

[0040] After the reaction is completed, a reaction solution containing cellooligosaccharides can be obtained. The reaction solution may contain enzymes such as SP and CDP, sucrose, and primers, and washing may be performed to remove these. Washing can be performed by repeating the steps of separating the precipitate and the supernatant by centrifugation and redispersing the precipitate using a washing solvent such as water, thereby obtaining cellooligosaccharides.

[0041] The obtained cellooligosaccharide is an oligosaccharide having a structure in which glucose is linked by β-1,4 glycosidic bonds. The degree of polymerization of the cellooligosaccharide may be, for example, 4 or more, 5 or more, or 6 or more, and may be, for example, 16 or less, 15 or less, or 13 or less. The average degree of polymerization (DP) is not particularly limited, and may be, for example, 5.5 or more, 6.0 or more, 6.5 or more, or 7.0 or more. The average degree of polymerization (DP) may also be, for example, 10.0 or less, 9.5 or less, or 9.0 or less.

[0042] An example of the obtained cellooligosaccharide is represented by the following general formula (3). [ka] In formula (3), R 8 is a hydroxy group, R 1 , or R 7 where n is an integer and indicates the degree of polymerization of cellooligosaccharide. The wavy line in the formula indicates that the anomeric configuration is α, β, or a mixture of α and β.

[0043] When glucose or cellobiose was used as a primer, R 8 is a hydroxy group. When the glucose derivative represented by the above formula (1) is used as a primer, R 8 is R in the above general formula (1). 1 When the cellobiose derivative represented by the above formula (2) is used as a primer, R 8 is R in the above general formula (2). 7 In this way, when a glucose derivative or a cellobiose derivative is used as a primer, a reducing end modified cellooligosaccharide in which the anomeric position of the reducing end is modified is obtained. The cellooligosaccharide according to this embodiment includes such cellooligosaccharide derivatives, and naturally also includes alkali metal ion adducts such as sodium ion adducts and potassium ion adducts. 8 After synthesizing cellooligosaccharides in which R is an alkoxy group, R is hydrolyzed to obtain 8It is also possible to produce cellooligosaccharides in which the is a hydroxy group.

[0044] The applications of the cellooligosaccharides according to this embodiment are not particularly limited, and they can be used for various known applications, such as in the pharmaceutical field. EXAMPLES

[0045] The present invention will be further described below with reference to examples, but is not limited thereto. 3 " and "mol / L" are abbreviated as "mM" and "M", respectively.

[0046] [reagent] In the present examples, unless otherwise specified, ultrapure water purified with a Milli-Q system (Milli-Q Advantage A-10, Merck Millipore) was used.

[0047] The 200 mM phosphate buffer was prepared by adding 0.2 M disodium hydrogen phosphate solution to 0.2 M sodium dihydrogen phosphate aqueous solution to adjust the pH to 7.4. Note that the initial concentration of phosphoric acid in this example is the sum of the concentrations of sodium dihydrogen phosphate and disodium hydrogen phosphate.

[0048] The 1M HEPES buffer solution was prepared by dissolving 23.8 g of 2-[4-(2-hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES) in 60 mL of water, adding 4N aqueous sodium hydroxide to the solution to adjust the pH to 7.5, and then diluting the solution to 100 mL with water.

[0049] The 20 mM MOPS buffer solution was prepared by dissolving 4.18 g of 3-morpholinopropanesulfonic acid (MOPS) in 600 mL of water, adding 4 N aqueous sodium hydroxide to the solution to adjust the pH to 7.5, then diluting the solution to 1 L with water and sterilizing it by filtration using a 0.22 μm PVDF filter.

[0050] The SP solution used was prepared by mixing sucrose phosphorylase (SP) powder (derived from Leuconostoc mesenteroides, manufactured by Oriental Yeast Co., Ltd.) with 20 mM MOPS buffer (pH 7.5) and adjusting the SP concentration to 10 U / mL.

[0051] CDP was prepared based on the method described in M. Krishnareddy et al., J. Appl. Glycosci., 2002, 49, 1-8. In detail, CDP was prepared by the method described in paragraphs 0061 to 0070 of Patent Document 1 (JP Patent Publication 2019-193601 A), and the obtained CDP was mixed with 20 mM MOPS buffer (pH 7.5) and adjusted to 10 U / mL for use.

[0052] ProteoMass TM Bradykinin fragment 1-7 MALDI-MS standard (Bradykinin), ProteoMass TM P 14 R MALDI-MS Standard (P 14 R), ProteoMass TM ACTH Fragment 18-39 MALDI-MS standard (ACTH), trifluoroacetic acid (TFA), and acetonitrile were purchased from Sigma-Aldrich.

[0053] Sucrose was purchased from Nacalai Tesque. Glucose, methyl glucoside (Me-GLC), and azidodeoxyglucoside (N3-dGLC) were used as primers. D-(+)-glucose was purchased from Nacalai Tesque as glucose. 1-methyl-α-D-glucopyranoside was purchased from Nacalai Tesque as methyl glucoside. 1-azido-1-deoxy-β-D-glucopyranoside was purchased from Sigma-Aldrich as azidodeoxyglucoside.

[0054] Unless otherwise specified, other reagents were purchased from Nacalai Tesque and were of special grade or higher.

[0055] [Examples 1 to 5, Comparative Example 1] A 5 mL reaction solution was prepared by adding 200 mM phosphate buffer, 1 M sucrose aqueous solution, 1 M glucose aqueous solution, SP solution, CDP solution and water to a 5 mL tube so that the concentrations of each component were as shown in Table 1 below. The pH of the reaction solution is shown in Table 1. The reaction solution was sealed with parafilm and incubated at 40°C for 3 days. The tube was then heated at 100°C for 5 minutes to inactivate the enzyme. The reaction solution was transferred to a 50 mL centrifuge tube, diluted to 20 mL with water, centrifuged (15000g) for 10 minutes, and purified by removing the supernatant. This purification procedure was repeated five times to obtain an aqueous dispersion of cellooligosaccharide. The obtained aqueous dispersion was dried at 70°C for 24 hours to obtain a dried cellooligosaccharide product, and the yield was measured.

[0056] The resulting product is a cellooligosaccharide represented by the following formula (4). [ka]

[0057] [Examples 6 and 7] Instead of glucose as a primer, methyl glucoside (Me-GLC) was used in Example 6, and azidodeoxyglucoside (N3-dGLC) was used in Example 7. Products were obtained and the yields were measured by carrying out the same procedures as in Example 2, except that the primer was replaced with methyl glucoside (Me-GLC) in Example 6 and azidodeoxyglucoside (N3-dGLC) in Example 7.

[0058] The resulting products were reducing end modified cellooligosaccharides represented by the following formula (5) in Example 6 and by the following formula (6) in Example 7. [ka]

[0059] [Example 8] A product was obtained by the same procedure as in Example 2, except that the incubation temperature was 30° C., and the yield was measured.

[0060] [Example 9] In Example 2, 0.25 mL of the water used in preparing the reaction solution was replaced with dimethyl sulfoxide (DMSO), and the solvent of the reaction solution was changed to a mixed solvent containing 5 volume % of DMSO. The same operations as in Example 2 were carried out to obtain a product, and the yield was measured.

[0061] [Example 10] In Example 2, 0.5 mL of the water used in preparing the reaction solution was replaced with DMSO, and the solvent of the reaction solution was changed to a mixed solvent containing 10 volume % DMSO. The same operations as in Example 2 were carried out, except that a product was obtained and the yield was measured.

[0062] [Example 11] The same procedure as in Example 2 was carried out except that 0.5 mL of the water used in preparing the reaction solution in Example 2 was replaced with methanol, and the solvent of the reaction solution was changed to a mixed solvent containing 10 volume % of methanol, to obtain a product, and the yield was measured.

[0063] [Example 12] The same procedure as in Example 2 was carried out except that 0.5 mL of the water used in preparing the reaction solution in Example 2 was replaced with N,N-dimethylformamide (DMF) and the solvent of the reaction solution was changed to a mixed solvent containing 10 volume % DMF, to obtain a product and measure the yield.

[0064] [Comparative Example 2] 1M HEPES buffer, 1M αG1P aqueous solution, 1M glucose aqueous solution, CDP solution and water were added to a 5mL tube so that the concentrations of each component were 500mM, 200mM, 50mM and 0.2U / mL, respectively, to prepare a 5mL reaction solution, which was then sealed with parafilm and incubated at 40°C for 3 days. The tube was then heated at 100°C for 5 minutes to inactivate the enzyme. The subsequent purification and drying steps were carried out in the same manner as in Example 1 to obtain a product, and the yield was measured.

[0065] The average degree of polymerization (DP), polymerization degree distribution, and yield of cellooligosaccharides were measured for the product obtained above. The results are shown in Table 1. The measurement methods are as follows.

[0066] (1) Average degree of polymerization The average degree of polymerization of cellooligosaccharides was measured by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

[0067] The standard samples used for mass-to-charge ratio calibration were Bradykin fragment 1-7 aqueous solution (10 nmol / mL Bradykinin fragment 1-7, 0.05% by mass trifluoroacetic acid (TFA), 50% by mass acetonitrile), P 14 R aqueous solution (10nmol / mL P 14 The solution was prepared by mixing 5 μL each of an ACTH fragment 18-39 aqueous solution (10 nmol / mL ACTH fragment 18-39, 0.1% by mass TFA), 5 μL of a 10 mg / mL DHBA aqueous solution, 1 μL of a 1.0% by mass TFA aqueous solution, and 4 μL of acetonitrile in a 1.7 mL tube.

[0068] The measurement sample of the product was prepared by mixing 1 μL of a 0.1% (w / v) aqueous dispersion of the product, 1 μL of a 10 mg / mL aqueous DHBA solution, and 3 μL of an acetonitrile solution of trifluoroacetic acid (0.2% by volume) in a mighty vial that had been washed with a methanol solution of potassium hydroxide (3.3% by mass).

[0069] 1 μL of each of the standard sample and the measurement sample of the product was mounted on a sample plate and air-dried five times. The sample was dried in vacuum for more than one hour and measured by MALDI-TOF-MS (AXIMA-performance, Shimadzu Corporation). The measurement conditions were Mode: Linear (positive), Mass Range: 1.0-3000.0, Max Leaser Rap Rate: 10, power: 100, profiles: 100, shots: 2, Ion Gate (Da): Blank 500, Pulsed Extraction optimized at (Da): 1000.0.

[0070] The MS spectra obtained by the measurements were processed under the following conditions: Smoothing method: Gaussian, Smoothing filter width: 19, Baseline filter width: 1000.

[0071] Since the measured sample contained sodium ion adducts and potassium ion adducts, the peak areas of these were summed to calculate the peak areas for each degree of polymerization. The average degree of polymerization was calculated from the ratio of the peak areas for each degree of polymerization thus calculated, and this was taken as the average degree of polymerization.

[0072] (2) Polymerization degree distribution The MALDI-TOF-MS spectrum obtained above was analyzed according to the following formula to determine the standard deviation from the average degree of polymerization, and the standard deviation was taken as the distribution of the degree of polymerization.

number

[0073] (3) Yield The yield was calculated from the yield of cellooligosaccharide (product) and the average degree of polymerization using the following formula, which was the conversion rate of the starting material, sucrose.

number

[0074] [Table 1]

[0075] The obtained product was confirmed to have a cellulose type II crystal structure by infrared spectroscopy (IR). According to Examples 1 to 12, the two-step synthesis yielded cellooligosaccharides similar to those obtained by the one-step synthesis in Comparative Example 2, and there was no significant difference in the average degree of polymerization.

[0076] On the other hand, in Examples 1 to 12, the yield of cellooligosaccharides was significantly improved compared to Comparative Example 2, which was a one-step synthesis. In particular, the yield was significantly higher when the phosphate concentrations were 10 mM and 50 mM compared to Examples 1 to 4. In contrast, in Comparative Example 1, in which the phosphate concentration exceeded 120 mM, the yield was low, and the one-step synthesis yield was only comparable to that of Comparative Example 2.

[0077] In Example 6, methyl glucoside was used as a primer, and it was confirmed by MALDI-TOF-MS that cellooligosaccharides with the reducing end substituted with a methoxy group were produced. In other words, reducing end-modified cellooligosaccharides could also be produced by the two-step synthesis. As shown in Table 1, it can be seen that such reducing end-modified cellooligosaccharides can be produced in the same yield and with the same degree of polymerization distribution as unmodified cellooligosaccharides.

[0078] In Example 7, azido-deoxyglucoside was used as a primer, and proton nuclear magnetic resonance ( 1 H-NMR measurements confirmed that the peak derived from the reducing end had disappeared compared to unmodified cellooligosaccharides. Fourier transform infrared spectroscopy (FT-IR) measurements also confirmed that the peak at 2120 cm -1 A peak derived from the azide group was confirmed around the peak. This confirmed that the reducing end was modified with an azide group. As shown in Table 1, the azide-added cellooligosaccharides were produced in the same yield and with the same degree of polymerization distribution as unmodified cellooligosaccharides.

[0079] Where:1 H-NMR and FT-IR were measured using a lyophilized product. 1 For H-NMR, a Bruker AV-400M was used, and approximately 15 mg of cellooligosaccharide was dissolved in 500 μL of 4% sodium deuterium hydroxide deuterium oxide solution, and the measurement was performed under the condition of 16 accumulations. For FT-IR, a Thermo Scientific Nicolet 6700 was used, and the ATR method was used with a resolution of 2 cm. -1 The measurements were performed under the condition of 32 cumulative measurements.

[0080] Examples 9 to 11 are examples in which a mixed solvent containing water and a water-soluble organic solvent was used. By using a mixed solvent in the two-stage synthesis reaction system, it was possible to narrow the polymerization degree distribution compared to Example 2.

[0081] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the gist of the invention. These embodiments and their omissions, substitutions, modifications, etc. are included in the scope and gist of the invention as well as the invention and its equivalents described in the claims.

Claims

1. The method includes a reaction step of reacting a primer in which a hydroxy group bonded to a carbon atom at the anomeric position of glucose or cellobiose is replaced by an organic group containing an azide group with sucrose phosphorylase and cellodextrin phosphorylase in the presence of phosphoric acid, and a phosphoric acid concentration in the reaction system, including the amount contained as an intermediate product, α-glucose-1-phosphate, of 8 mol / m ３ More than 70mol / m ３ The method for producing cellooligosaccharides is as follows.

2. The method for producing cellooligosaccharides according to claim 1, wherein the reaction temperature is 25°C or higher and 45°C or lower.

3. 3. The method for producing cellooligosaccharides according to claim 1, wherein the primer and the sucrose are reacted with the sucrose phosphorylase and the cellodextrin phosphorylase in a mixed solvent containing water and a water-soluble organic solvent.

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