WO2014199948A1 - Production method for β-mannoside - Google Patents

Abstract

　Provided is a simple and inexpensive method for producing β-mannoside. This method for producing β-mannoside is characterized in that, in the presence of phosphoric acid, α-phosphoglucomutase (EC 5.4.2.2), glucose-6-phosphate isomerase (EC 5.3.1.9), mannose-6-phosphate isomerase (EC 5.3.1.8), α-phosphomannomutase (EC 5.4.2.8), and cofactors thereof, (i) a combination of a carbohydrate starting material and an enzyme capable of reversible phosphorolysis of the carbohydrate starting material to form α-glucose-1-phosphoric acid, and (ii) a combination of an enzyme capable of reversible phosphorolysis of β-mannoside to form α-mannose-1-phosphoric acid and a substance acting as a sugar acceptor in a reverse reaction thereof, are caused to act.

Description

Process for producing β-mannoside

The present invention relates to a method for producing β-mannoside using an enzymatic method.

Sugar chains are said to be the third chain after nucleic acids and proteins. Recently, the importance of biorecognition (cell adhesion, antigen-antibody reaction, information transmission, virus infection, etc.) has attracted attention. It is being advanced. Among them, β-mannoside, the core structure of asparagine-linked sugar chains and lipopolysaccharides, is deeply involved in life phenomena such as cell differentiation, aging, and immune response, and diseases such as cancer, virus infection, and inflammation. Elucidation of the function of sugar chains at the molecular level, and further application to the sugar chain medical industry as glycoprotein preparations, immunostimulators, pathogenic virus infection inhibitors and the like are expected.

Preparation of β-mannoside with a very small amount of expression in vivo must rely on an organic synthesis method that currently requires a complicated multi-step reaction (Patent Document 1), and efficient mass preparation is difficult. The problem of being extremely expensive has hindered the progress of the glycoengineering research field and the sugar chain medical industry.

It has been suggested that β-mannoside can be synthesized using α-mannose-1-phosphate as a raw material by utilizing the reverse reaction catalytic activity of an enzyme that reversibly phosphorylates β-mannoside ( Patent Documents 2 and 3, Non-Patent Document 1) and α-mannose-1-phosphate are expensive, and thus lacked practicality due to cost problems.

Japanese Patent No. 4778315 Japanese Patent Application No. 2012-190474 Japanese Patent Application No. 2012-203891

Therefore, an object is to provide a method for producing β-mannoside at low cost and with ease.

As a result of intensive studies to achieve the above-mentioned problems, the present inventors have completed a method for producing β-mannoside at low cost by an enzymatic method.

That is, the present invention includes the following.
(1) Phosphate, α-phosphoglucomutase (EC 5.4.2.2), glucose-6-phosphate isomerase (EC 5.3.1.9), mannose-6-phosphate isomerase (EC 5 3.3.1.8), α-phosphomannomutase (EC 5.4.2.8) and their cofactors,
(I) a carbohydrate raw material, and a combination of enzymes that reversibly phosphorolyze the carbohydrate raw material to produce α-glucose-1-phosphate; and (ii) reversibly phosphorolytically decompose β-mannoside And a method for producing β-mannoside, wherein a combination of an enzyme that produces α-mannose-1-phosphate and a substance that acts as a sugar acceptor in the reverse reaction is allowed to act.

(2) The saccharide material of (i) and the combination of enzymes that reversibly phosphorolyze the saccharide material to produce α-glucose-1-phosphate are sucrose and sucrose phosphorylase (EC 2.4. 1.7), starch or dextrin and phosphorylase (EC 2.4.1.1), cellobiose and cellobiose phosphorylase (EC 2.4.1.20), cellodextrin and cellodextrin Combination with phosphorylase (EC 2.4.4.19) and cellobiose phosphorylase (EC 2.4.1.20), laminari oligosaccharide and laminaribiose phosphorylase (EC 2.4.1.31) and / or β- Combination with 1,3 oligoglucan phosphorylase (EC 2.4.1.30), sophoro-oligosaccharide and sono One or more combinations selected from the group consisting of a combination of phorose phosphorylase and / or β-1,2 oligoglucan phosphorylase, and a combination of trehalose and trehalose phosphorylase (EC 2.4.1.231) The method according to (1) above.

(3) The combination of an enzyme that reversibly phosphorylates β-mannoside in (ii) to produce α-mannose-1-phosphate and a substance that acts as a sugar acceptor in the reverse reaction, A combination of 1,4-N-acetylglucosamine phosphorylase and N-acetylglucosamine and / or N, N′-diacetylchitobiose, a combination of β-1,2-mannobiose phosphorylase and mannose and / or fructose, Selected from the group consisting of a combination of mannosyl-β-1,4-glucose phosphorylase and glucose, and a combination of β-1,4-mannooligosaccharide phosphorylase and mannose and / or β-1,4-mannooligosaccharide The method according to (1) above, which is a combination of one or more.

(4) The method according to any one of (1) to (3) above, wherein the enzyme is immobilized on a carrier.

According to the present invention, there is provided an inexpensive and simple method for producing β-mannoside.

FIG. 1 shows a schematic diagram of the production of mannosyl-β-1,4-N-acetylglucosamine from sucrose. FIG. 2 shows a schematic diagram of the production of β-1,2-mannobiose from sucrose. FIG. 3 shows a schematic diagram of the production of mannosyl-β-1,4-glucose from sucrose. FIG. 4 shows a schematic diagram of the production of mannosyl-β-1,4-N-acetylglucosamine from cellobiose. FIG. 5 shows a schematic diagram of the production of β-1,2-mannobiose from cellobiose. FIG. 6 shows a schematic diagram of the production of mannosyl-β-1,4-glucose from cellobiose. FIG. 7 shows a schematic diagram of the production of mannosyl-β-1,4-N-acetylglucosamine from starch. FIG. 8 shows a schematic diagram of the production of β-1,2-mannobiose from starch. FIG. 9 shows a schematic diagram of the production of mannosyl-β-1,4-glucose from starch.

The present invention relates to a method for producing β-mannoside by an enzymatic method.

The enzyme method of the present invention mainly comprises the following three enzyme reactions: (1) Phospholysis reaction of saccharide raw material; (2) α-mannose-1-phosphoric acid of α-glucose-1-phosphate (3) Synthesis reaction of β-mannoside from sugar acceptor.

The phosphorolysis reaction of the saccharide raw material in (1) includes the saccharide raw material contained in the element (i) and an enzyme that generates a α-glucose-1-phosphate by phosphorolysis of the saccharide raw material ( (G1P-producing enzyme) is a reaction in which α-glucose-1-phosphate and reducing end sugar are produced by reaction in the presence of phosphoric acid. The combination of the enzyme with the carbohydrate raw material used in the element (i) is not limited to this, but a combination of sucrose and sucrose phosphorylase, a combination of starch or dextrin and phosphorylase, cellobiose and cellobiose phosphorylase, A combination of cellodextrin and cellodextrin phosphorylase and cellobiose phosphorylase, a combination of laminary oligosaccharide and laminaribiose phosphorylase and / or β-1,3 oligoglucan phosphorylase, sophoro-oligosaccharide and sophorose phosphorylase and / or β-1 , 2 A combination of oligoglucan phosphorylase, a combination of trehalose and trehalose phosphorylase, or a combination of two or more thereof. More preferred combinations are one of a combination of sucrose and sucrose phosphorylase, a combination of cellobiose and cellobiose phosphorylase, a combination of cellodextrin and cellodextrin phosphorylase and cellobiose phosphorylase, a combination of starch or dextrin and phosphorylase, or two The most preferred combination is a combination of sucrose and sucrose phosphorylase.

The concentration of the sugar raw material used is not particularly limited, but is preferably about 1 to about 1000 g / L, and more preferably about 10 to about 1000 g / L.

As the G1P-producing enzyme, any source enzyme can be used, and its use form is not particularly limited, and various substances such as bacterial cell extract, purified enzyme, and immobilized enzyme can be used. The amount used is also not particularly limited, and for example, it can be used at about 0.1 mg to about 1000 mg per 1 g of the saccharide raw material.

Moreover, the phosphoric acid involved in this reaction may be of any origin. The concentration of phosphoric acid added to the reaction system is not particularly limited, but is preferably about 0.1 mM to about 1000 mM, more preferably about 1 mM to about 100 mM.

The conversion reaction of α-glucose-1-phosphate to α-mannose-1-phosphate in the above (2) includes α-phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, α-Glucose-1-phosphate generated by the phosphorolysis reaction of the saccharide raw material of (1) above is converted into glucose 6-phosphate, glucose- In this reaction, 6-phosphate is converted to fructose-6-phosphate, fructose-6-phosphate is converted to mannose-6-phosphate, and mannose-6-phosphate is converted to α-mannose-1-phosphate.

These enzymes are not particularly limited, and enzymes of any origin can be used. The usage form of this enzyme is not particularly limited, and various substances such as bacterial cell extract, purified enzyme, and immobilized enzyme can be used, and the amount of the enzyme used (represented by activity) is not particularly limited. Can be used, for example, at about 1 mg to about 1000 mg per gram of carbohydrate raw material. The use concentration of the cofactor is not particularly limited, but is preferably about 0.01 mM to about 100 mM, more preferably about 0.02 mM to about 50 mM.

The synthesis reaction of β-mannoside from the sugar acceptor described in (3) above is an enzyme (β-mannoside phosphorylase) that reversibly phosphorylates β-mannoside to produce α-mannose-1-phosphate. In the presence of a sugar acceptor as a starting material, α-mannose-1-phosphate produced by the conversion reaction of α-glucose-1-phosphate to α-mannose-1-phosphate in the above (2) is converted into β -Reactions that synthesize mannosides. The concentration of the sugar acceptor used as a starting material is not particularly limited, but is preferably about 10 mM to about 2M, more preferably about 100 mM to about 1M. β-Mannoside phosphorylase is not particularly limited, and an enzyme of any origin can be used. Preferably, mannosyl-β-1,4-N-acetylglucosamine phosphorylase, β-1,2-mannobiose phosphorylase, mannosyl-β-1,4-glucose phosphorylase, β-1,4-mannooligosaccharide phosphorylase is used. The usage form of β-mannoside phosphorylase is not particularly limited, and various substances such as a bacterial cell extract, purified enzyme, and immobilized enzyme can be used. The amount of β-mannoside phosphorylase to be used is not particularly limited. For example, it can be used in an amount of about 0.1 mg to about 1000 mg per 1 g of the saccharide raw material.

The above-described enzyme of the present invention may be of any origin, for example, any enzyme derived from prokaryotes such as bacteria, eukaryotes such as yeast, fungi, and animals, and is a recombinant enzyme. May be. Such enzymes may be commercially available, or may be purified by methods well known to those skilled in the art, for example, purified from nature, or obtained by genetic recombination methods. For example, in the method by gene recombination, the enzyme of the present invention is PCR using a primer prepared based on the nucleotide sequence of the enzyme gene described in the literature or registered in a known nucleic acid or protein sequence database. By amplifying a cDNA prepared from mRNA corresponding to the enzyme gene in a suitable library by incorporating the cDNA into a commercially available gene expression vector, and transforming cells such as E. coli with the expression vector, It is produced in the fungus body. The produced enzyme can be purified by protein purification methods well known to those skilled in the art, such as crude fraction such as ammonium sulfate fraction or various column chromatography. Further, the subsequent purification can be facilitated by expressing the enzyme as a fusion protein with GST or His-tag.

The enzyme of the present invention may also be directly purified from the above prokaryotic cells or eukaryotic cells. Prepare cell lysate, centrifuge, ammonium sulfate fractionation, dialysis, various chromatography (eg gel filtration chromatography, ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, etc.), electrophoresis, ultrafiltration The target enzyme can be purified by appropriately combining general techniques for enzyme purification such as crystallization. The form of the enzyme that can be used in the present invention may be a crude enzyme (for example, microbial cell extract, lyophilized product, etc.) in addition to the purified enzyme. If a crude enzyme is used, it should not contain factors that interfere with the above reaction of the present invention.

The reaction form is not particularly limited, but it is usually performed in an aqueous solution or a buffer solution. The pH of the reaction solution is preferably 5-9. The reaction temperature is not particularly limited, but is preferably 5 ° C to 80 ° C, more preferably 20 ° C to 60 ° C. The reaction time is not particularly limited, but is preferably 0.1 to 3000 hours.

One advantage of the present invention is that all the enzyme reactions described above can be carried out simply and easily in one container or using a bioreactor.

The β-mannoside obtained by the present invention can be purified by any method. For example, β-mannoside obtained by the present invention can be isolated by column chromatography or crystallization. Examples of column chromatography include, but are not limited to, size exclusion chromatography, silica gel column chromatography, ion exchange chromatography, ultrafiltration membrane separation, and reverse osmosis membrane separation. Crystallization methods include, but are not limited to, concentration, temperature reduction, and solvent addition (ethanol, methanol, acetone, etc.).

The present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the spirit of the present invention.

Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.

A conversion reaction to mannosyl-β-1,4-N-acetylglucosamine was performed using sucrose as a carbohydrate raw material. The reaction volume is 1 mL, and the final concentration consists of 0.25 M sucrose, 0.25 M N-acetylglucosamine, 10 mM magnesium chloride, 25 mM phosphate-sodium buffer (pH 7.0), 59 μM glucose-1,6-bisphosphate. A substrate solution is prepared, and sucrose phosphorylase, mannosyl-β-1,4-N-acetylglucosamine phosphorylase, α-phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, α-phospho Mannomutase was added at 0.033 mg, 0.083 mg, 0.34 mg, 0.37 mg, 0.23 mg, and 2.4 mg per milliliter, and reacted at 30 ° C. for 120 hours. The reaction was stopped by adding 0.13 mL of 6N hydrochloric acid, and after adjusting the pH of the reaction solution to 4.5, the invertase treatment was performed to decompose residual sucrose. The reaction solution was applied to a TOYOPEARL HW-40S column, and the mannosyl-β-1,4-N-acetylglucosamine fraction was isolated and collected by gel filtration. The collected fraction was concentrated by an evaporator and freeze-dried to obtain 20 mg of mannosyl-β-1,4-N-acetylglucosamine preparation.

A conversion reaction to β-1,2-mannobiose was performed using sucrose as a carbohydrate raw material. The reaction volume is 1 mL, and a substrate solution consisting of a final concentration of 0.25 M sucrose, 0.25 M mannose, 10 mM magnesium chloride, 25 mM phosphate-sodium buffer (pH 7.0), 59 μM glucose-1,6-bisphosphate is used. 1 milliliter of sucrose phosphorylase, β-1,2-mannobiose phosphorylase, α-phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, α-phosphomannometase 0.033 mg, 0.050 mg, 0.34 mg, 0.37 mg, 0.23 mg, and 2.4 mg were added and reacted at 30 ° C. for 120 hours. The reaction was stopped by adding 0.13 mL of 6N hydrochloric acid, and after adjusting the pH of the reaction solution to 4.5, the invertase treatment was performed to decompose residual sucrose. The reaction solution was applied to a TOYOPEARL HW-40S column, and β-1,2-mannobiose fraction was isolated and collected by gel filtration. The collected fraction was concentrated by an evaporator and freeze-dried to obtain 39 mg of β-1,2-mannobiose sample.

Conversion reaction to mannosyl-β-1,4-glucose was performed using sucrose as a saccharide raw material. The reaction volume is 1 mL, and a substrate solution consisting of a final concentration of 0.25 M sucrose, 10 mM magnesium chloride, 25 mM phosphate-sodium buffer (pH 7.0), 59 μM glucose-1,6-bisphosphate is prepared. Sucrose phosphorylase, mannosyl-β-1,4-glucose phosphorylase, α-phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, α-phosphomannomutase, xylose isomerase is 0 per milliliter. 0.33 mg, 0.76 mg, 0.34 mg, 0.37 mg, 0.23 mg, 2.4 mg, and 0.33 mg were added, and the reaction was performed at 30 ° C. for 120 hours. The reaction was stopped by adding 0.13 mL of 6N hydrochloric acid, and after adjusting the pH of the reaction solution to 4.5, the invertase treatment was performed to decompose residual sucrose. The reaction solution was applied to a TOYOPEARL HW-40S column, and the mannosyl-β-1,4-glucose fraction was isolated and collected by gel filtration. The collected fraction was concentrated with an evaporator and lyophilized to obtain 28 mg of mannosyl-β-1,4-glucose preparation.

Conversion reaction to mannosyl-β-1,4-N-acetylglucosamine was performed using cellobiose as a carbohydrate raw material. The reaction volume is 1 mL, and the final concentration is 0.25 M cellobiose, 0.25 M N-acetylglucosamine, 10 mM magnesium chloride, 25 mM phosphate-sodium buffer (pH 7.0), 59 μM glucose-1,6-bisphosphate. A substrate solution is prepared, and cellobiose phosphorylase, mannosyl-β-1,4-N-acetylglucosamine phosphorylase, α-phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, α-phospho Mannomutase was added at 2.3 mg, 0.083 mg, 0.34 mg, 0.37 mg, 0.23 mg, and 2.4 mg per milliliter, and reacted at 30 ° C. for 120 hours. The reaction was stopped by adding 0.13 mL of 6N hydrochloric acid, and after adjusting the pH of the reaction solution to 5, the remaining cellobiose was decomposed by treatment with β-glucosidase. The reaction solution was applied to a TOYOPEARL HW-40S column, and the mannosyl-β-1,4-N-acetylglucosamine fraction was isolated and collected by gel filtration. The collected fraction was concentrated by an evaporator and freeze-dried to obtain 12 mg of mannosyl-β-1,4-N-acetylglucosamine preparation.

Conversion reaction to β-1,2-mannobiose was performed using cellobiose as a carbohydrate raw material. The reaction volume is 1 mL, and a substrate solution consisting of a final concentration of 0.25 M cellobiose, 0.25 M mannose, 10 mM magnesium chloride, 25 mM phosphate-sodium buffer (pH 7.0), 59 μM glucose-1,6-bisphosphate is used. 1 ml of cellobiose phosphorylase, β-1,2-mannobiose phosphorylase, α-phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, α-phosphomannometase 2.3 mg, 0.050 mg, 0.34 mg, 0.37 mg, 0.23 mg, and 2.4 mg were added, and the reaction was performed at 30 ° C. for 120 hours. The reaction was stopped by adding 0.13 mL of 6N hydrochloric acid, and after adjusting the pH of the reaction solution to 5, the remaining cellobiose was decomposed by treatment with β-glucosidase. The reaction solution was applied to a TOYOPEARL HW-40S column, and β-1,2-mannobiose fraction was isolated and collected by gel filtration. The collected fraction was concentrated by an evaporator and freeze-dried to obtain 15 mg of β-1,2-mannobiose sample.

Conversion reaction to mannosyl-β-1,4-glucose was performed using cellobiose as a saccharide raw material. The reaction volume is 1 mL, and a substrate solution consisting of a final concentration of 0.25 M cellobiose, 10 mM magnesium chloride, 25 mM phosphate-sodium buffer (pH 7.0), 59 μM glucose-1,6-bisphosphate is prepared. 0.083 mg per milliliter of cellobiose phosphorylase, mannosyl-β-1,4-glucose phosphorylase, α-phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, 0.033 mg, 0.34 mg, 0.37 mg, 0.23 mg, and 2.4 mg were added, and the reaction was performed at 30 ° C. for 120 hours. The reaction was stopped by adding 0.13 mL of 6N hydrochloric acid, and after adjusting the pH of the reaction solution to 5, the remaining cellobiose was decomposed by treatment with β-glucosidase. The reaction solution was applied to a TOYOPEARL HW-40S column, and the mannosyl-β-1,4-glucose fraction was isolated and collected by gel filtration. The collected fraction was concentrated with an evaporator and freeze-dried to obtain 46 mg of mannosyl-β-1,4-glucose preparation.

The starch was converted into mannosyl-β-1,4-N-acetylglucosamine using starch as a saccharide raw material. The reaction volume is 1 mL, consisting of a final concentration of 50 mg / mL starch, 0.25 M N-acetylglucosamine, 10 mM magnesium chloride, 25 mM phosphate-sodium buffer (pH 7.0), 59 μM glucose-1,6-bisphosphate. A substrate solution was prepared, and phosphorylase, mannosyl-β-1,4-N-acetylglucosamine phosphorylase, α-phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, α-phosphoman 0.96 mg, 0.083 mg, 0.34 mg, 0.37 mg, 0.23 mg, 2.4 mg, and 0.3 μg of nomutase and isoamylase were added per milliliter, and the reaction was performed at 30 ° C. for 120 hours. The reaction was stopped by adding 0.13 mL of 6N hydrochloric acid, and after adjusting the pH of the reaction solution to 4.5, the residual starch was decomposed by glucoamylase treatment. The reaction solution was applied to a TOYOPEARL HW-40S column, and the mannosyl-β-1,4-N-acetylglucosamine fraction was isolated and collected by gel filtration. The collected fraction was concentrated with an evaporator, and 6 mg of mannosyl-β-1,4-N-acetylglucosamine preparation was obtained by freeze-drying.

デ ン プ ン Conversion reaction to β-1,2-mannobiose was performed using starch as a saccharide material. The reaction volume is 1 mL, and a substrate solution consisting of a final concentration of 50 mg / mL starch, 0.25 M mannose, 10 mM magnesium chloride, 25 mM phosphate-sodium buffer (pH 7.0), 59 μM glucose-1,6-bisphosphate is used. Prepared, phosphorylase, β-1,2-mannobiose phosphorylase, α-phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, α-phosphomannomutase, isomerase 0.96 mg, 0.050 mg, 0.34 mg, 0.37 mg, 0.23 mg, and 2.4 mg were added per milliliter, and the reaction was performed at 30 ° C. for 120 hours. The reaction was stopped by adding 0.13 mL of 6N hydrochloric acid, and after adjusting the pH of the reaction solution to 4.5, the residual starch was decomposed by glucoamylase treatment. The reaction solution was applied to a TOYOPEARL HW-40S column, and β-1,2-mannobiose fraction was isolated and collected by gel filtration. The collected fraction was concentrated with an evaporator and freeze-dried to obtain 11 mg of β-1,2-mannobiose preparation.

The starch was converted into mannosyl-β-1,4-glucose as a saccharide raw material. The reaction volume is 1 mL, and a substrate solution consisting of a final concentration of 50 mg / mL starch, 0.25 M glucose, 10 mM magnesium chloride, 25 mM phosphate-sodium buffer (pH 7.0), 59 μM glucose-1,6-bisphosphate is used. Prepared, phosphorylase, nnosyl-β-1,4-glucose phosphorylase, α-phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, α-phosphomannomutase, isoamylase 0.96 mg, 0.76 mg, 0.34 mg, 0.37 mg, 0.23 mg, 2.4 mg, and 0.33 μg were added per milliliter, and the reaction was performed at 30 ° C. for 120 hours. The reaction was stopped by adding 0.13 mL of 6N hydrochloric acid, and after adjusting the pH of the reaction solution to 4.5, the residual starch was decomposed by glucoamylase treatment. The reaction solution was applied to a TOYOPEARL HW-40S column, and the mannosyl-β-1,4-glucose fraction was isolated and collected by gel filtration. The collected fraction was concentrated by an evaporator and lyophilized to obtain 11 mg of mannosyl-β-1,4-glucose preparation.

As described above, the present invention can be used in the pharmaceutical, food and research reagent industries.

Claims (3)

1. Phosphate, α-phosphoglucomutase (EC 5.4.2.2), glucose-6-phosphate isomerase (EC 5.3.1.9), mannose-6-phosphate isomerase (EC 5.3. 1.8), in the presence of α-phosphomannomutase (EC 5.4.2.8) and their cofactors,
   (I) a carbohydrate raw material, and a combination of enzymes that reversibly phosphorolyze the carbohydrate raw material to produce α-glucose-1-phosphate; and (ii) reversibly phosphorolytically decompose β-mannoside And a method for producing β-mannoside, wherein a combination of an enzyme that produces α-mannose-1-phosphate and a substance that acts as a sugar acceptor in the reverse reaction is allowed to act.
2. The combination of the saccharide raw material (i) and the enzyme that reversibly phosphorylates the saccharide raw material to produce α-glucose-1-phosphate is sucrose and sucrose phosphorylase (EC 2.4.1.7). ), Starch or dextrin and phosphorylase (EC 2.4.1.1), cellobiose and cellobiose phosphorylase (EC 2.4.1.20), cellodextrin and cellodextrin phosphorylase (EC) 2.4.4.19) and cellobiose phosphorylase (EC 2.4.2.10), laminari oligosaccharides and laminaribiose phosphorylase (EC 2.4.1.31) and / or β-1,3 Combination with oligoglucan phosphorylase (EC 2.4.1.30), sophoro-oligosaccharide and soholo One or more combinations selected from the group consisting of a combination of sphosphorylase and / or β-1,2 oligoglucan phosphorylase, and a combination of trehalose and trehalose phosphorylase (EC 2.4.1.231) The method of claim 1.
3. A combination of an enzyme that reversibly phosphorylates β-mannoside in (ii) to produce α-mannose-1-phosphate, and a substance that acts as a sugar acceptor in the reverse reaction, is mannosyl-β-1,4. A combination of N-acetylglucosamine phosphorylase with N-acetylglucosamine and / or N, N′-diacetylchitobiose, a combination of β-1,2-mannobiose phosphorylase with mannose and / or fructose, mannosyl-β One or more selected from the group consisting of a combination of -1,4-glucose phosphorylase and glucose, and a combination of β-1,4-mannooligosaccharide phosphorylase and mannose and / or β-1,4-mannooligosaccharide The method of claim 1, which is a combination of:

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