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WO2014199948A1 - Production method for β-mannoside - Google Patents

Production method for β-mannoside Download PDF

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Publication number
WO2014199948A1
WO2014199948A1 PCT/JP2014/065223 JP2014065223W WO2014199948A1 WO 2014199948 A1 WO2014199948 A1 WO 2014199948A1 JP 2014065223 W JP2014065223 W JP 2014065223W WO 2014199948 A1 WO2014199948 A1 WO 2014199948A1
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Prior art keywords
phosphorylase
combination
glucose
phosphate
mannose
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PCT/JP2014/065223
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French (fr)
Japanese (ja)
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博之 中井
高則 仁平
大坪　研一
北岡　本光
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国立大学法人新潟大学
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    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/12—Disaccharides
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose

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  • the present invention relates to a method for producing ⁇ -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, ⁇ -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.
  • ⁇ -mannoside can be synthesized using ⁇ -mannose-1-phosphate as a raw material by utilizing the reverse reaction catalytic activity of an enzyme that reversibly phosphorylates ⁇ -mannoside ( Patent Documents 2 and 3, Non-Patent Document 1) and ⁇ -mannose-1-phosphate are expensive, and thus lacked practicality due to cost problems.
  • an object is to provide a method for producing ⁇ -mannoside at low cost and with ease.
  • the present inventors have completed a method for producing ⁇ -mannoside at low cost by an enzymatic method.
  • the present invention includes the following. (1) Phosphate, ⁇ -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), ⁇ -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 ⁇ -glucose-1-phosphate; and (ii) reversibly phosphorolytically decompose ⁇ -mannoside And a method for producing ⁇ -mannoside, wherein a combination of an enzyme that produces ⁇ -mannose-1-phosphate and a substance that acts as a sugar acceptor in the reverse reaction is allowed to act.
  • the saccharide material of (i) and the combination of enzymes that reversibly phosphorolyze the saccharide material to produce ⁇ -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 ⁇ - Combination with 1,3 oligoglucan phosphorylase (EC 2.4.1.30), sophoro-oligosaccharide and sono
  • FIG. 1 shows a schematic diagram of the production of mannosyl- ⁇ -1,4-N-acetylglucosamine from sucrose.
  • FIG. 2 shows a schematic diagram of the production of ⁇ -1,2-mannobiose from sucrose.
  • FIG. 3 shows a schematic diagram of the production of mannosyl- ⁇ -1,4-glucose from sucrose.
  • FIG. 4 shows a schematic diagram of the production of mannosyl- ⁇ -1,4-N-acetylglucosamine from cellobiose.
  • FIG. 5 shows a schematic diagram of the production of ⁇ -1,2-mannobiose from cellobiose.
  • FIG. 6 shows a schematic diagram of the production of mannosyl- ⁇ -1,4-glucose from cellobiose.
  • FIG. 7 shows a schematic diagram of the production of mannosyl- ⁇ -1,4-N-acetylglucosamine from starch.
  • FIG. 8 shows a schematic diagram of the production of ⁇ -1,2-mannobiose from starch.
  • FIG. 9 shows a schematic diagram of the production of mannosyl- ⁇ -1,4-glucose from starch.
  • the present invention relates to a method for producing ⁇ -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) ⁇ -mannose-1-phosphoric acid of ⁇ -glucose-1-phosphate (3) Synthesis reaction of ⁇ -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 ⁇ -glucose-1-phosphate by phosphorolysis of the saccharide raw material ( (G1P-producing enzyme) is a reaction in which ⁇ -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 ⁇ -1,3 oligoglucan phosphorylase, sophoro-oligosaccharide and sophorose phosphorylase and / or ⁇ -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.
  • 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.
  • the phosphoric acid involved in this reaction may be of any origin.
  • 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 ⁇ -glucose-1-phosphate to ⁇ -mannose-1-phosphate in the above (2) includes ⁇ -phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, ⁇ -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 ⁇ -mannose-1-phosphate.
  • 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 ⁇ -mannoside from the sugar acceptor described in (3) above is an enzyme ( ⁇ -mannoside phosphorylase) that reversibly phosphorylates ⁇ -mannoside to produce ⁇ -mannose-1-phosphate.
  • ⁇ -mannoside phosphorylase enzyme that reversibly phosphorylates ⁇ -mannoside to produce ⁇ -mannose-1-phosphate.
  • ⁇ -mannose-1-phosphate produced by the conversion reaction of ⁇ -glucose-1-phosphate to ⁇ -mannose-1-phosphate in the above (2) is converted into ⁇ -Reactions that synthesize mannosides.
  • 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.
  • ⁇ -Mannoside phosphorylase is not particularly limited, and an enzyme of any origin can be used.
  • mannosyl- ⁇ -1,4-N-acetylglucosamine phosphorylase, ⁇ -1,2-mannobiose phosphorylase, mannosyl- ⁇ -1,4-glucose phosphorylase, ⁇ -1,4-mannooligosaccharide phosphorylase is used.
  • the usage form of ⁇ -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 ⁇ -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.
  • 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.
  • a cDNA prepared from mRNA corresponding to the enzyme gene in a suitable library 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 ⁇ -mannoside obtained by the present invention can be purified by any method.
  • ⁇ -mannoside obtained by the present invention can be isolated by column chromatography or crystallization.
  • 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.).
  • a conversion reaction to mannosyl- ⁇ -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 ⁇ M glucose-1,6-bisphosphate.
  • a substrate solution is prepared, and sucrose phosphorylase, mannosyl- ⁇ -1,4-N-acetylglucosamine phosphorylase, ⁇ -phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, ⁇ -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- ⁇ -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- ⁇ -1,4-N-acetylglucosamine preparation.
  • a conversion reaction to ⁇ -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 ⁇ M glucose-1,6-bisphosphate is used.
  • sucrose phosphorylase 1 milliliter of sucrose phosphorylase, ⁇ -1,2-mannobiose phosphorylase, ⁇ -phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, ⁇ -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.
  • reaction solution was applied to a TOYOPEARL HW-40S column, and ⁇ -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 ⁇ -1,2-mannobiose sample.
  • Conversion reaction to mannosyl- ⁇ -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 ⁇ M glucose-1,6-bisphosphate is prepared.
  • Sucrose phosphorylase, mannosyl- ⁇ -1,4-glucose phosphorylase, ⁇ -phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, ⁇ -phosphomannomutase, xylose isomerase is 0 per milliliter.
  • Conversion reaction to mannosyl- ⁇ -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 ⁇ M glucose-1,6-bisphosphate.
  • a substrate solution is prepared, and cellobiose phosphorylase, mannosyl- ⁇ -1,4-N-acetylglucosamine phosphorylase, ⁇ -phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, ⁇ -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 ⁇ -glucosidase.
  • the reaction solution was applied to a TOYOPEARL HW-40S column, and the mannosyl- ⁇ -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- ⁇ -1,4-N-acetylglucosamine preparation.
  • Conversion reaction to ⁇ -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 ⁇ M glucose-1,6-bisphosphate is used.
  • reaction solution was applied to a TOYOPEARL HW-40S column, and ⁇ -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 ⁇ -1,2-mannobiose sample.
  • Conversion reaction to mannosyl- ⁇ -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 ⁇ M glucose-1,6-bisphosphate is prepared.
  • the reaction solution was applied to a TOYOPEARL HW-40S column, and the mannosyl- ⁇ -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- ⁇ -1,4-glucose preparation.
  • the starch was converted into mannosyl- ⁇ -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 ⁇ M glucose-1,6-bisphosphate.
  • a substrate solution was prepared, and phosphorylase, mannosyl- ⁇ -1,4-N-acetylglucosamine phosphorylase, ⁇ -phosphoglucomutase, glucose-6-phosphate isomerase, mannose-6-phosphate isomerase, ⁇ -phosphoman 0.96 mg, 0.083 mg, 0.34 mg, 0.37 mg, 0.23 mg, 2.4 mg, and 0.3 ⁇ g 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- ⁇ -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- ⁇ -1,4-N-acetylglucosamine preparation was obtained by freeze-drying.
  • ⁇ ⁇ ⁇ ⁇ Conversion reaction to ⁇ -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 ⁇ M glucose-1,6-bisphosphate is used.
  • reaction solution was applied to a TOYOPEARL HW-40S column, and ⁇ -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 ⁇ -1,2-mannobiose preparation.
  • the starch was converted into mannosyl- ⁇ -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 ⁇ M glucose-1,6-bisphosphate is used.
  • the reaction solution was applied to a TOYOPEARL HW-40S column, and the mannosyl- ⁇ -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- ⁇ -1,4-glucose preparation.
  • the present invention can be used in the pharmaceutical, food and research reagent industries.

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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

β－マンノシドの製造方法Process for producing β-mannoside
　本発明は、酵素法を用いてβ－マンノシドを製造する方法に関する。 The present invention relates to a method for producing β-mannoside using an enzymatic method.
　糖鎖は、核酸、タンパク質に次ぐ第三の鎖といわれ、生体認識（細胞接着・抗原抗体反応・情報伝達・ウイルス感染など）の重要性について近年注目を集めており、急速にその機能解明が進められている。その中で、アスパラギン結合型糖鎖やリポ多糖のコア構造であるβ－マンノシドは、細胞分化、老化、免疫応答といった生命現象や、癌、ウイルス感染、炎症などの疾患に深く関与していることが知られており、分子レベルでの糖鎖機能の解明、さらには糖タンパク質製剤・免疫賦活剤・病原性ウイルス感染阻害剤などとしての糖鎖医療産業界への応用が期待されている。 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.
　生体内での発現量が微量なβ－マンノシドの調製は、現在煩雑な多段階反応を要する有機合成法に頼らざるを得ず（特許文献１）、効率的な大量調製が困難であるため、非常に高額であるという問題点から糖鎖工学研究分野や糖鎖医療産業の進展を妨げている。 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.
　β－マンノシドを可逆的に加リン酸分解する酵素の逆反応触媒活性を利用し、α－マンノース－１－リン酸を原料として、β－マンノシドを合成可能であることが示唆されているが（特許文献２及び３、非特許文献１）、α－マンノース－１－リン酸が高価であるため、コストの問題から実用性を欠いていた。 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.
特許第４７７８３１５号明細書Japanese Patent No. 4778315 特願２０１２－１９０４７４号明細書Japanese Patent Application No. 2012-190474 特願２０１２－２０３８９１号明細書Japanese Patent Application No. 2012-203891
　したがって、安価でかつ簡便にβ－マンノシドを製造する方法を提供することを目的とする。 Therefore, an object is to provide a method for producing β-mannoside at low cost and with ease.
　本発明者らは、上記課題を達成するため鋭意検討した結果、酵素法によりβ－マンノシドを安価に製造する方法を完成させた。 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.
　すなわち、本発明は以下を包含する。
　（１）リン酸、α－ホスホグルコムターゼ（ＥＣ　５．４．２．２）、グルコース－６－リン酸イソメラーゼ（ＥＣ　５．３．１．９）、マンノース－６－リン酸イソメラーゼ（ＥＣ　５．３．１．８）、α－ホスホマンノムターゼ（ＥＣ　５．４．２．８）及びそれらの補因子の存在下で、
　（ｉ）糖質原料、及び該糖質原料を可逆的に加リン酸分解しα－グルコース－１－リン酸を生じる酵素の組合せ；並びに
　（ｉｉ）β－マンノシドを可逆的に加リン酸分解してα－マンノース－１－リン酸を生じる酵素及びその逆反応において糖アクセプターとして作用する物質の組合せを作用させることを特徴とする、β－マンノシドの製造方法。 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）（ｉ）の糖質原料、及び該糖質原料を可逆的に加リン酸分解しα－グルコース－１－リン酸を生じる酵素の組合せが、スクロースとスクロースホスホリラーゼ（ＥＣ　２．４．１．７）との組合せ、デンプン若しくはデキストリンとホスホリラーゼ（ＥＣ　２．４．１．１）との組合せ、セロビオースとセロビオースホスホリラーゼ（ＥＣ　２．４．１．２０）との組合せ、セロデキストリンとセロデキストリンホスホリラーゼ（ＥＣ　２．４．１．４９）及びセロビオースホスホリラーゼ（ＥＣ　２．４．１．２０）との組合せ、ラミナリオリゴ糖とラミナリビオースホスホリラーゼ（ＥＣ　２．４．１．３１）及び／若しくはβ－１，３オリゴグルカンホスホリラーゼ（ＥＣ　２．４．１．３０）との組合せ、ソホロオリゴ糖とソホロースホスホリラーゼ及び／若しくはβ－１，２オリゴグルカンホスホリラーゼとの組合せ、並びにトレハロースとトレハロースホスホリラーゼ（ＥＣ　２．４．１．２３１）との組合せ、よりなる群から選択される１つ以上の組合せである、上記（１）に記載の方法。 (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）（ｉｉ）のβ－マンノシドを可逆的に加リン酸分解してα－マンノース－１－リン酸を生じる酵素及びその逆反応において糖アクセプターとして作用する物質の組合せが、マンノシル－β－１，４－Ｎ－アセチルグルコサミンホスホリラーゼとＮ－アセチルグルコサミン及び／若しくはＮ，Ｎ’－ジアセチルキトビオースとの組合せ、β－１，２－マンノビオースホスホリラーゼとマンノース及び／若しくはフルクトースとの組合せ、マンノシル－β－１，４－グルコースホスホリラーゼとグルコースとの組合せ、並びにβ－１，４－マンノオリゴ糖ホスホリラーゼとマンノース及び／若しくはβ－１，４－マンノオリゴ糖との組合せ、よりなる群から選択される１つ以上の組合せである、上記（１）に記載の方法。 (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）酵素が担体に固定化されている、上記（１）～（３）のいずれかに記載の方法。 (4) The method according to any one of (1) to (3) above, wherein the enzyme is immobilized on a carrier.
　本発明によれば、安価でかつ簡便にβ－マンノシドを製造する方法が提供される。 According to the present invention, there is provided an inexpensive and simple method for producing β-mannoside.
図１は、スクロースからのマンノシル－β－１,４－Ｎ－アセチルグルコサミンの製造の概略図を示す。FIG. 1 shows a schematic diagram of the production of mannosyl-β-1,4-N-acetylglucosamine from sucrose. 図２は、スクロースからのβ－１,２－マンノビオースの製造の概略図を示す。FIG. 2 shows a schematic diagram of the production of β-1,2-mannobiose from sucrose. 図３は、スクロースからのマンノシル－β－１，４－グルコースの製造の概略図を示す。FIG. 3 shows a schematic diagram of the production of mannosyl-β-1,4-glucose from sucrose. 図４は、セロビオースからのマンノシル－β－１,４－Ｎ－アセチルグルコサミンの製造の概略図を示す。FIG. 4 shows a schematic diagram of the production of mannosyl-β-1,4-N-acetylglucosamine from cellobiose. 図５は、セロビオースからのβ－１,２－マンノビオースの製造の概略図を示す。FIG. 5 shows a schematic diagram of the production of β-1,2-mannobiose from cellobiose. 図６は、セロビオースからのマンノシル－β－１，４－グルコースの製造の概略図を示す。FIG. 6 shows a schematic diagram of the production of mannosyl-β-1,4-glucose from cellobiose. 図７は、デンプンからのマンノシル－β－１,４－Ｎ－アセチルグルコサミンの製造の概略図を示す。FIG. 7 shows a schematic diagram of the production of mannosyl-β-1,4-N-acetylglucosamine from starch. 図８は、デンプンからのβ－１,２－マンノビオースの製造の概略図を示す。FIG. 8 shows a schematic diagram of the production of β-1,2-mannobiose from starch. 図９は、デンプンからのマンノシル－β－１，４－グルコースの製造の概略図を示す。FIG. 9 shows a schematic diagram of the production of mannosyl-β-1,4-glucose from starch.
　本発明は、酵素法による、β－マンノシドの製造方法に関する。 The present invention relates to a method for producing β-mannoside by an enzymatic method.
　本発明の酵素法は、主に以下の３つの酵素反応からなる：（１）糖質原料の加リン酸分解反応；（２）α－グルコース－１－リン酸のα－マンノース－１－リン酸への変換反応、（３）糖アクセプターからのβ－マンノシドの合成反応。 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.
　（１）の糖質原料の加リン酸分解反応は、上記要素（ｉ）に含まれる糖質原料と該糖質原料を加リン酸分解してα－グルコース－１－リン酸を生じる酵素（Ｇ１Ｐ生成酵素）との組合せが、リン酸の存在下で反応して、α－グルコース－１－リン酸及び還元末糖が生じる反応である。上記要素（ｉ）で用いられる糖質原料と係る酵素の組合せは、これに限定されるものではないが、スクロースとスクロースホスホリラーゼとの組合せ、デンプン若しくはデキストリンとホスホリラーゼとの組合せ、セロビオースとセロビオースホスホリラーゼとの組合せ、セロデキストリンとセロデキストリンホスホリラーゼ及びセロビオースホスホリラーゼとの組合せ、ラミナリオリゴ糖とラミナリビオースホスホリラーゼ及び／若しくはβ－１，３オリゴグルカンホスホリラーゼとの組合せ、ソホロオリゴ糖とソホロースホスホリラーゼ及び／若しくはβ－１，２オリゴグルカンホスホリラーゼとの組合せ、トレハロースとトレハロースホスホリラーゼとの組合せのいずれか一つ、あるいは二つ以上の組合せを含む。より好ましい組合せは、スクロースとスクロースホスホリラーゼとの組合せ、セロビオースとセロビオースホスホリラーゼとの組合せ、セロデキストリンとセロデキストリンホスホリラーゼ及びセロビオースホスホリラーゼとの組合せ、デンプン又はデキストリンとホスホリラーゼとの組合せのいずれか一つ、あるいは二つ以上の組合せであり、最も好ましい組合せは、スクロースとスクロースホスホリラーゼとの組合せである。 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.
　糖質原料の使用濃度は特に限定されるものではないが、好ましくは約１～約１０００ｇ／Ｌであり、より好ましくは約１０～約１０００ｇ／Ｌである。 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.
　Ｇ１Ｐ生成酵素はいかなる起源の酵素を用いることも可能であり、その使用形態は特に限定されるものではなく、菌体抽出液、精製酵素、固定化酵素など種々のものを利用することができ、その使用量も特に限定されないが、例えば糖質原料１ｇあたり約０．１ｍｇ～約１０００ｍｇで使用し得る。 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.
　またこの反応に関わるリン酸はいかなる起源のものであっても良い。反応系に加えるリン酸濃度は特に限定されるものではないが、好ましくは約０．１ｍＭ～約１０００ｍＭ、より好ましくは約１ｍＭ～約１００ｍＭ程度である。 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.
　上記（２）のα－グルコース－１－リン酸のα－マンノース－１－リン酸への変換反応は、α－ホスホグルコムターゼ、グルコース－６－リン酸イソメラーゼ、マンノース－６－リン酸イソメラーゼ、α－ホスホマンノムターゼ及びそれらの補因子の組合せによって、上記（１）の糖質原料の加リン酸分解反応で生じたα－グルコース－１－リン酸をグルコース６－リン酸に、グルコース－６－リン酸をフルクトース－６－リン酸に、フルクトース－６－リン酸をマンノース－６－リン酸に、マンノース－６－リン酸をα－マンノース－１－リン酸へ変換する反応である。 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.
　これらの酵素は特に限定されるものではなくいかなる起源の酵素を用いることも可能である。この酵素の使用形態は特に限定されるものではなく、菌体抽出液、精製酵素、固定化酵素など種々のものを利用することができ、その酵素の使用量（活性で表す）も特に限定されないが、例えば糖質原料１ｇあたり約１ｍｇ～約１０００ｍｇで使用し得る。補因子の使用濃度は特に限定されるものではないが、好ましくは約０．０１ｍＭ～約１００ｍＭ、より好ましくは約０．０２ｍＭ～約５０ｍＭである。 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.
　上記（３）の糖アクセプターからのβ－マンノシドの合成反応は、β－マンノシドを可逆的に加リン酸分解してα－マンノース－１－リン酸を生じる酵素（β－マンノシドホスホリラーゼ）の存在下で、糖アクセプターを出発原料として、上記（２）のα－グルコース－１－リン酸のα－マンノース－１－リン酸への変換反応によって生じたα－マンノース－１－リン酸からβ－マンノシドを合成する反応である。出発原料として用いる糖アクセプターの濃度は特に限定されないが、好ましくは約１０ｍＭ～約２Ｍ、より好ましくは約１００ｍＭ～約１Ｍである。β－マンノシドホスホリラーゼは特に限定されるものではなく、またいかなる起源の酵素を用いることも可能である。好ましくはマンノシル－β－１，４－Ｎ－アセチルグルコサミンホスホリラーゼ、β－１，２－マンノビオースホスホリラーゼ、マンノシル－β－１，４－グルコースホスホリラーゼ、β－１，４－マンノオリゴ糖ホスホリラーゼを用いる。β－マンノシドホスホリラーゼの使用形態は特に限定されるものではなく、菌体抽出液、精製酵素、固定化酵素など種々のものを利用することができる。β－マンノシドホスホリラーゼの使用量も特に限定されないが、例えば糖質原料１ｇあたり約０．１ｍｇ～約１０００ｍｇで使用し得る。 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.
　上述した本発明の酵素は、任意の起源のものでよく、例えば細菌等の原核生物、酵母、菌類、動物等の真核生物由来のいずれの酵素であってもよく、また組換え酵素であってもよい。そのような酵素は市販のものを使用し得るか、または当業者に周知の方法、例えば天然から精製してもよいし、あるいは遺伝子組換え法によって取得し得る。例えば、遺伝子組換えによる方法では、本発明の酵素は、文献に記載されている若しくは公知の核酸又はタンパク質配列データベースに登録されている該酵素遺伝子の塩基配列を基に作製したプライマーを用いたＰＣＲによって適当なライブラリー中の該酵素遺伝子に対応するｍＲＮＡから作製したｃＤＮＡを増幅した後に、該ｃＤＮＡを市販の遺伝子発現ベクターに組込み、該発現ベクターで大腸菌等の菌体を形質転換することによって、菌体中で生成される。生成された酵素は、硫安分画等の粗分画又は各種のカラムクロマトグラフィーなど、当業者に周知のタンパク質精製法によって精製できる。また、酵素をＧＳＴやＨｉｓ－ｔａｇとの融合タンパク質として発現させることにより、その後の精製を容易にすることができる。 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.
　本発明の酵素はまた、上記の原核生物細胞又は真核生物細胞から直接精製してもよい。細胞破壊液を調製し、遠心分離、硫安分画、透析、各種クロマトグラフィー（例えばゲル濾過クロマトグラフィー、イオン交換クロマトグラフィー、疎水性相互作用クロマトグラフィー、アフィニティクロマトグラフィーなど）、電気泳動、限外ろ過、結晶化などの酵素精製のための一般的な技術を適宜組合せて、目的の酵素を精製することができる。本発明に使用可能な酵素の形態は、精製酵素の他に、粗製酵素（例えば菌体抽出液、凍結乾燥体など）でもよい。粗製酵素を使用する場合には、本発明の上記反応を妨害する因子を含むべきではない。 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.
　反応形態は特に限定されるものではないが、通常は水溶液又は緩衝液中で行われる。反応液のｐＨは好ましくは５～９である。反応温度は特に限定されるものではないが、好ましくは５℃～８０℃、より好ましくは２０℃～６０℃である。また反応時間は特に限定されるものではないが、０．１～３０００時間であることが好ましい。 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.
　本発明の一つの利点は、上記全ての酵素反応を、一容器中で又はバイオリアクターを用いて簡便かつ容易に実施できる点にある。 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.
　本発明により得られるβ－マンノシドは任意の方法で精製することができる。例えば、本発明により得られるβ－マンノシドは、カラムクロマトグラフィーや結晶化により単離することが可能である。カラムクロマトグラフィーとして、これに限定されるものではないが、サイズ排除クロマトグラフィー、シリカゲルカラムクロマトグラフィー、イオン交換クロマトグラフィー、限外濾過膜分離、逆浸透膜分離が含まれる。結晶化方法としては、これに限定されるものではないが、濃縮、温度低下、溶媒添加（エタノール、メタノール、アセトンなど）が含まれる。 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.).
　なお、本発明は上記実施形態に限定されるものではなく、本発明の思想を逸脱しない範囲で種々の変形実施が可能である。 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.
　次に、本発明を実施例により詳しく説明するが、本発明はこれらにより限定されるものではない。 Next, the present invention will be described in detail with reference to examples, but the present invention is not limited thereto.
　スクロースを糖質原料としてマンノシル－β－１，４－Ｎ－アセチルグルコサミンへの変換反応を行った。反応液量は１ｍＬとし、終濃度０．２５Ｍ　スクロース、０．２５Ｍ　Ｎ－アセチルグルコサミン、１０ｍＭ　塩化マグネシウム、２５ｍＭ　リン酸－ナトリウム緩衝液（ｐＨ７．０）、５９μＭ　グルコース－１，６－ビスリン酸からなる基質溶液を調製し、そこにスクロースホスホリラーゼ、マンノシル－β－１，４－Ｎ－アセチルグルコサミンホスホリラーゼ、α－ホスホグルコムターゼ、グルコース－６－リン酸イソメラーゼ、マンノース－６－リン酸イソメラーゼ、α－ホスホマンノムターゼを１ミリリットル当たり０．０３３ｍｇ、０．０８３ｍｇ、０．３４ｍｇ、０．３７ｍｇ、０．２３ｍｇ、２．４ｍｇ加え、３０℃で１２０時間反応を行った。反応を０．１３ｍＬの６Ｎ　塩酸を添加することで反応を停止し、反応液のｐＨを４．５に調整後、インベルターゼ処理を行うことにより残存スクロースを分解した。反応液をＴＯＹＯＰＥＡＲＬ　ＨＷ－４０Ｓカラムに供し、ゲル濾過によりマンノシル－β－１，４－Ｎ－アセチルグルコサミン画分を単離、回収した。回収した画分をエバポレーターで濃縮し、凍結乾燥によりマンノシル－β－１，４－Ｎ－アセチルグルコサミン標品２０ｍｇを得た。 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.
　スクロースを糖質原料としてβ－１，２－マンノビオースへの変換反応を行った。反応液量は１ｍＬとし、終濃度０．２５Ｍ　スクロース、０．２５Ｍ　マンノース、１０ｍＭ　塩化マグネシウム、２５ｍＭ　リン酸－ナトリウム緩衝液（ｐＨ７．０）、５９μＭ　グルコース－１，６－ビスリン酸からなる基質溶液を調製し、そこにスクロースホスホリラーゼ、β－１，２－マンノビオースホスホリラーゼ、α－ホスホグルコムターゼ、グルコース－６－リン酸イソメラーゼ、マンノース－６－リン酸イソメラーゼ、α－ホスホマンノムターゼを１ミリリットル当たり０．０３３ｍｇ、０．０５０ｍｇ、０．３４ｍｇ、０．３７ｍｇ、０．２３ｍｇ、２．４ｍｇ加え、３０℃で１２０時間反応を行った。反応を０．１３ｍＬの６Ｎ　塩酸を添加することで反応を停止し、反応液のｐＨを４．５に調整後、インベルターゼ処理を行うことにより残存スクロースを分解した。反応液をＴＯＹＯＰＥＡＲＬ　ＨＷ－４０Ｓカラムに供し、ゲル濾過によりβ－１，２－マンノビオース画分を単離、回収した。回収した画分をエバポレーターで濃縮し、凍結乾燥によりβ－１，２－マンノビオース標品３９ｍｇを得た。 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.
　スクロースを糖質原料としてマンノシル－β－１，４－グルコースへの変換反応を行った。反応液量は１ｍＬとし、終濃度０．２５Ｍ　スクロース、１０ｍＭ　塩化マグネシウム、２５ｍＭ　リン酸－ナトリウム緩衝液（ｐＨ７．０）、５９μＭ　グルコース－１，６－ビスリン酸からなる基質溶液を調製し、そこにスクロースホスホリラーゼ、マンノシル－β－１，４－グルコースホスホリラーゼ、α－ホスホグルコムターゼ、グルコース－６－リン酸イソメラーゼ、マンノース－６－リン酸イソメラーゼ、α－ホスホマンノムターゼ、キシロースイソメラーゼを１ミリリットル当たり０．０．３３ｍｇ、０．７６ｍｇ、０．３４ｍｇ、０．３７ｍｇ、０．２３ｍｇ、２．４ｍｇ、０．３３ｍｇ加え、３０℃で１２０時間反応を行った。反応を０．１３ｍＬの６Ｎ　塩酸を添加することで反応を停止し、反応液のｐＨを４．５に調整後、インベルターゼ処理を行うことにより残存スクロースを分解した。反応液をＴＯＹＯＰＥＡＲＬ　ＨＷ－４０Ｓカラムに供し、ゲル濾過によりマンノシル－β－１，４－グルコース画分を単離、回収した。回収した画分をエバポレーターで濃縮し、凍結乾燥によりマンノシル－β－１，４－グルコース標品２８ｍｇを得た。 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.
　セロビオースを糖質原料としてマンノシル－β－１，４－Ｎ－アセチルグルコサミンへの変換反応を行った。反応液量は１ｍＬとし、終濃度０．２５Ｍ　セロビオース、０．２５Ｍ　Ｎ－アセチルグルコサミン、１０ｍＭ　塩化マグネシウム、２５ｍＭ　リン酸－ナトリウム緩衝液（ｐＨ７．０）、５９μＭ　グルコース－１，６－ビスリン酸からなる基質溶液を調製し、そこにセロビオースホスホリラーゼ、マンノシル－β－１，４－Ｎ－アセチルグルコサミンホスホリラーゼ、α－ホスホグルコムターゼ、グルコース－６－リン酸イソメラーゼ、マンノース－６－リン酸イソメラーゼ、α－ホスホマンノムターゼを１ミリリットル当たり２．３ｍｇ、０．０８３ｍｇ、０．３４ｍｇ、０．３７ｍｇ、０．２３ｍｇ、２．４ｍｇ加え、３０℃で１２０時間反応を行った。反応を０．１３ｍＬの６Ｎ　塩酸を添加することで反応を停止し、反応液のｐＨを５に調整後、β－グルコシダーゼ処理を行うことにより残存セロビオースを分解した。反応液をＴＯＹＯＰＥＡＲＬ　ＨＷ－４０Ｓカラムに供し、ゲル濾過によりマンノシル－β－１，４－Ｎ－アセチルグルコサミン画分を単離、回収した。回収した画分をエバポレーターで濃縮し、凍結乾燥によりマンノシル－β－１，４－Ｎ－アセチルグルコサミン標品１２ｍｇを得た。 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.
　セロビオースを糖質原料としてβ－１，２－マンノビオースへの変換反応を行った。反応液量は１ｍＬとし、終濃度０．２５Ｍ　セロビオース、０．２５Ｍ　マンノース、１０ｍＭ　塩化マグネシウム、２５ｍＭ　リン酸－ナトリウム緩衝液（ｐＨ７．０）、５９μＭ　グルコース－１，６－ビスリン酸からなる基質溶液を調製し、そこにセロビオースホスホリラーゼ、β－１，２－マンノビオースホスホリラーゼ、α－ホスホグルコムターゼ、グルコース－６－リン酸イソメラーゼ、マンノース－６－リン酸イソメラーゼ、α－ホスホマンノムターゼを１ミリリットル当たり２．３ｍｇ、０．０５０ｍｇ、０．３４ｍｇ、０．３７ｍｇ、０．２３ｍｇ、２．４ｍｇ加え、３０℃で１２０時間反応を行った。反応を０．１３ｍＬの６Ｎ　塩酸を添加することで反応を停止し、反応液のｐＨを５に調整後、β－グルコシダーゼ処理を行うことにより残存セロビオースを分解した。反応液をＴＯＹＯＰＥＡＲＬ　ＨＷ－４０Ｓカラムに供し、ゲル濾過によりβ－１，２－マンノビオース画分を単離、回収した。回収した画分をエバポレーターで濃縮し、凍結乾燥によりβ－１，２－マンノビオース標品１５ｍｇを得た。 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.
　セロビオースを糖質原料としてマンノシル－β－１，４－グルコースへの変換反応を行った。反応液量は１ｍＬとし、終濃度０．２５Ｍ　セロビオース、１０ｍＭ　塩化マグネシウム、２５ｍＭ　リン酸－ナトリウム緩衝液（ｐＨ７．０）、５９μＭ　グルコース－１，６－ビスリン酸からなる基質溶液を調製し、そこにセロビオースホスホリラーゼ、マンノシル－β－１，４－グルコースホスホリラーゼ、α－ホスホグルコムターゼ、グルコース－６－リン酸イソメラーゼ、マンノース－６－リン酸イソメラーゼ、α－ホスホマンノムターゼを１ミリリットル当たり０．０８３ｍｇ、０．０３３ｍｇ、０．３４ｍｇ、０．３７ｍｇ、０．２３ｍｇ、２．４ｍｇ加え、３０℃で１２０時間反応を行った。反応を０．１３ｍＬの６Ｎ　塩酸を添加することで反応を停止し、反応液のｐＨを５に調整後、β－グルコシダーゼ処理を行うことにより残存セロビオースを分解した。反応液をＴＯＹＯＰＥＡＲＬ　ＨＷ－４０Ｓカラムに供し、ゲル濾過によりマンノシル－β－１，４－グルコース画分を単離、回収した。回収した画分をエバポレーターで濃縮し、凍結乾燥によりマンノシル－β－１，４－グルコース標品４６ｍｇを得た。 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.
　デンプンを糖質原料としてマンノシル－β－１，４－Ｎ－アセチルグルコサミンへの変換反応を行った。反応液量は１ｍＬとし、終濃度５０ｍｇ／ｍＬ　デンプン、０．２５Ｍ　Ｎ－アセチルグルコサミン、１０ｍＭ　塩化マグネシウム、２５ｍＭ　リン酸－ナトリウム緩衝液（ｐＨ７．０）、５９μＭ　グルコース－１，６－ビスリン酸からなる基質溶液を調製し、そこにホスホリラーゼ、マンノシル－β－１，４－Ｎ－アセチルグルコサミンホスホリラーゼ、α－ホスホグルコムターゼ、グルコース－６－リン酸イソメラーゼ、マンノース－６－リン酸イソメラーゼ、α－ホスホマンノムターゼ、イソアミラーゼを１ミリリットル当たり０．９６ｍｇ、０．０８３ｍｇ、０．３４ｍｇ、０．３７ｍｇ、０．２３ｍｇ、２．４ｍｇ、０．３μｇ加え、３０℃で１２０時間反応を行った。反応を０．１３ｍＬの６Ｎ　塩酸を添加することで反応を停止し、反応液のｐＨを４．５に調整後、グルコアミラーゼ処理を行うことにより残存デンプンを分解した。反応液をＴＯＹＯＰＥＡＲＬ　ＨＷ－４０Ｓカラムに供し、ゲル濾過によりマンノシル－β－１，４－Ｎ－アセチルグルコサミン画分を単離、回収した。回収した画分をエバポレーターで濃縮し、凍結乾燥によりマンノシル－β－１，４－Ｎ－アセチルグルコサミン標品６ｍｇを得た。 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.
　デンプンを糖質原料としてβ－１，２－マンノビオースへの変換反応を行った。反応液量は１ｍＬとし、終濃度５０ｍｇ／ｍＬ　デンプン、０．２５Ｍ　マンノース、１０ｍＭ　塩化マグネシウム、２５ｍＭ　リン酸－ナトリウム緩衝液（ｐＨ７．０）、５９μＭ　グルコース－１，６－ビスリン酸からなる基質溶液を調製し、そこにホスホリラーゼ、β－１，２－マンノビオースホスホリラーゼ、α－ホスホグルコムターゼ、グルコース－６－リン酸イソメラーゼ、マンノース－６－リン酸イソメラーゼ、α－ホスホマンノムターゼ、イソアミラーゼを１ミリリットル当たり０．９６ｍｇ、０．０５０ｍｇ、０．３４ｍｇ、０．３７ｍｇ、０．２３ｍｇ、２．４ｍｇ加え、３０℃で１２０時間反応を行った。反応を０．１３ｍＬの６Ｎ　塩酸を添加することで反応を停止し、反応液のｐＨを４．５に調整後、グルコアミラーゼ処理を行うことにより残存デンプンを分解した。反応液をＴＯＹＯＰＥＡＲＬ　ＨＷ－４０Ｓカラムに供し、ゲル濾過によりβ－１，２－マンノビオース画分を単離、回収した。回収した画分をエバポレーターで濃縮し、凍結乾燥によりβ－１，２－マンノビオース標品１１ｍｇを得た。 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.
　デンプンを糖質原料としてマンノシル－β－１，４－グルコースへの変換反応を行った。反応液量は１ｍＬとし、終濃度５０ｍｇ／ｍＬ　デンプン、０．２５Ｍ　グルコース、１０ｍＭ　塩化マグネシウム、２５ｍＭ　リン酸－ナトリウム緩衝液（ｐＨ７．０）、５９μＭ　グルコース－１，６－ビスリン酸からなる基質溶液を調製し、そこにホスホリラーゼ、ンノシル－β－１，４－グルコースホスホリラーゼ、α－ホスホグルコムターゼ、グルコース－６－リン酸イソメラーゼ、マンノース－６－リン酸イソメラーゼ、α－ホスホマンノムターゼ、イソアミラーゼを１ミリリットル当たり０．９６ｍｇ、０．７６ｍｇ、０．３４ｍｇ、０．３７ｍｇ、０．２３ｍｇ、２．４ｍｇ、０．３３μｇ加え、３０℃で１２０時間反応を行った。反応を０．１３ｍＬの６Ｎ　塩酸を添加することで反応を停止し、反応液のｐＨを４．５に調整後、グルコアミラーゼ処理を行うことにより残存デンプンを分解した。反応液をＴＯＹＯＰＥＡＲＬ　ＨＷ－４０Ｓカラムに供し、ゲル濾過によりマンノシル－β－１，４－グルコース画分を単離、回収した。回収した画分をエバポレーターで濃縮し、凍結乾燥によりマンノシル－β－１，４－グルコース標品１１ｍｇを得た。 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.
　以上のように本発明は、医薬品・食品・研究試薬産業で利用できる。 As described above, the present invention can be used in the pharmaceutical, food and research reagent industries.

Claims (3)

  1. 　リン酸、α－ホスホグルコムターゼ（ＥＣ　５．４．２．２）、グルコース－６－リン酸イソメラーゼ（ＥＣ　５．３．１．９）、マンノース－６－リン酸イソメラーゼ（ＥＣ　５．３．１．８）、α－ホスホマンノムターゼ（ＥＣ　５．４．２．８）及びそれらの補因子の存在下で、
    　（ｉ）糖質原料、及び該糖質原料を可逆的に加リン酸分解しα－グルコース－１－リン酸を生じる酵素の組合せ；並びに
    　（ｉｉ）β－マンノシドを可逆的に加リン酸分解してα－マンノース－１－リン酸を生じる酵素及びその逆反応において糖アクセプターとして作用する物質の組合せを作用させることを特徴とする、β－マンノシドの製造方法。     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. 　（ｉ）の糖質原料、及び該糖質原料を可逆的に加リン酸分解しα－グルコース－１－リン酸を生じる酵素の組合せが、スクロースとスクロースホスホリラーゼ（ＥＣ　２．４．１．７）との組合せ、デンプン若しくはデキストリンとホスホリラーゼ（ＥＣ　２．４．１．１）との組合せ、セロビオースとセロビオースホスホリラーゼ（ＥＣ　２．４．１．２０）との組合せ、セロデキストリンとセロデキストリンホスホリラーゼ（ＥＣ　２．４．１．４９）及びセロビオースホスホリラーゼ（ＥＣ　２．４．１．２０）との組合せ、ラミナリオリゴ糖とラミナリビオースホスホリラーゼ（ＥＣ　２．４．１．３１）及び／若しくはβ－１，３オリゴグルカンホスホリラーゼ（ＥＣ　２．４．１．３０）との組合せ、ソホロオリゴ糖とソホロースホスホリラーゼ及び／若しくはβ－１，２オリゴグルカンホスホリラーゼとの組合せ、並びにトレハロースとトレハロースホスホリラーゼ（ＥＣ　２．４．１．２３１）との組合せ、よりなる群から選択される１つ以上の組合せである、請求項１に記載の方法。 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. 　（ｉｉ）のβ－マンノシドを可逆的に加リン酸分解してα－マンノース－１－リン酸を生じる酵素及びその逆反応において糖アクセプターとして作用する物質の組合せが、マンノシル－β－１，４－Ｎ－アセチルグルコサミンホスホリラーゼとＮ－アセチルグルコサミン及び／若しくはＮ，Ｎ’－ジアセチルキトビオースとの組合せ、β－１，２－マンノビオースホスホリラーゼとマンノース及び／若しくはフルクトースとの組合せ、マンノシル－β－１，４－グルコースホスホリラーゼとグルコースとの組合せ、並びにβ－１，４－マンノオリゴ糖ホスホリラーゼとマンノース及び／若しくはβ－１，４－マンノオリゴ糖との組合せ、よりなる群から選択される１つ以上の組合せである、請求項１に記載の方法。 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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