WO2003062442A1 - PROCESS FOR PRODUCING PYRIDOXINE-5’-α-GLUCOSIDE - Google Patents

PROCESS FOR PRODUCING PYRIDOXINE-5’-α-GLUCOSIDE Download PDF

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WO2003062442A1
WO2003062442A1 PCT/JP2003/000543 JP0300543W WO03062442A1 WO 2003062442 A1 WO2003062442 A1 WO 2003062442A1 JP 0300543 W JP0300543 W JP 0300543W WO 03062442 A1 WO03062442 A1 WO 03062442A1
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pyridoxine
genus
salt
boric acid
producing
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PCT/JP2003/000543
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French (fr)
Japanese (ja)
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Koichi Wada
Keiji Sakamoto
Yasuhisa Asano
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Daiichi Fine Chemical Co., Ltd.
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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
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/58—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound through only acyclic carbon atoms to a non-saccharide heterocyclic ring, e.g. bleomycin, phleomycin
    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring

Definitions

  • the present invention relates to a method for producing pyridoxine-1 5′- ⁇ _darcoside, and more particularly, to the ability to selectively glycosylate pyridoxine at the 5-position to produce pyridoxine-15,1- ⁇ -darcoside.
  • the present invention also relates to a method for producing pyridoxine mono- ⁇ -darcoside by treating pyridoxine or a salt thereof with a glycosidase, the method of enhancing selectivity for glycosylation at the 5-position.
  • Pyridoxine ( ⁇ ) is one of the substances that has vitamin B6 action together with pyridoxal (PL) and pyridoxamine (PM).
  • PN taken into cells is phosphorylated to form pyridoxine-15, monophosphate (PNP), which is further oxidized to pyridoxal-5'-phosphate (PLP), an enzyme that participates in amino acid metabolism. It plays an important role as a coenzyme.
  • Pyridoxine hydrochloride (PN.HC1) is soluble in water but is unstable to light, and has problems of acidity and bitterness.
  • pyridoxine- ⁇ -darcoside has excellent photostability [J. Vitaminol., 17, 121-124 (1971)], and has no or less acidity or bitterness.
  • PN.HC1 Pyridoxine hydrochloride
  • Pyridoxine-1 ⁇ -darcoside has glycosides at positions 5 and 4 ', but pyridoxine-1-5,1- ⁇ -darcoside is rapidly hydrolyzed to ⁇ by homogenized hepatocytes. Or Therefore, they are highly useful as pharmaceuticals and health foods, and mass production is desired.
  • a method for producing pyridoxine-15, _ ⁇ -darcoside using a microorganism cell or an enzyme produced therefrom a method using a microorganism of the genus Micrococcus [Japanese Patent Publication No. 46-33198 (1971)] , J. Vitaminology 15, 142-150 (1969),
  • an object of the present invention is to provide a method for selectively glycosylating the 5′-position of pyridoxine. More specifically, a microbial strain capable of producing pyridoxine-15,1- ⁇ -darcoside was found, and pyridoxine or a salt thereof was obtained by using the cells, cultures, or processed cells of the microorganism. An object of the present invention is to provide a method for easily and efficiently producing only pyridoxine 15, 1 ⁇ -glucoside from glycerol.
  • Another object of the present invention is to provide a method for producing pyridoxine mono-Dalcoside by treating pyridoxine or a salt thereof with a glycosylase, which enhances the selectivity of glycosylation at the 5-position. It is in.
  • the present researchers have screened a wide variety of highly selective strains for microorganisms whose genus species have been identified to solve the above-mentioned problems. As a result, the selectivity among bacteria and fungi was 5, Especially high microorganisms were found. Further, pyridoxine or a salt thereof is When producing pyridoxine-hydarcoside by treating with a bacterial cell, a culture thereof, a treated bacterial cell, or a glycosidase, the treatment is performed in the presence of boric acid and / or borate And that the selectivity for glycosylation at the 5th position is enhanced. The present invention has been completed based on these findings.
  • the present invention relates to a method for producing pyridoxine-5, -a-darcoside by glycosylation of the 5′-position of pyridoxine, which belongs to any one of the following genera.
  • a method comprising the step of treating pyridoxine or a salt thereof with a microorganism, a culture thereof, or a treated product of a microorganism capable of selectively glycosylating and producing pyridoxine-1,5-a-darcoside. Is provided.
  • the above method wherein the treatment is performed in the presence of boric acid and / or borate.
  • the present invention provides a method for producing pyridoxine mono-darcoside by treating pyridoxine or a salt thereof with a glycoside enzyme, wherein boric acid and / or borate is used.
  • the method of treating pyridoxine or a salt thereof with glycosidase to produce pyridoxine mono- ⁇ -darcoside is carried out in the presence of
  • the present invention provides a reagent for enhancing the glycosylation selectivity, which reagent comprises boric acid and ⁇ ⁇ or borate.
  • pyridoxine or a dalcoside is produced by using pyridoxine or a salt thereof as a starting compound and selectively glycosylating the 5-position of pyridoxine to form pyridoxine.
  • pyridoxine or a salt thereof is produced by using pyridoxine or a salt thereof as a starting compound and selectively glycosylating the 5-position of pyridoxine to form pyridoxine.
  • ' It is characterized by using bacterial cells of microorganisms capable of producing ⁇ -darcoside, cultures thereof, or processed cells.
  • microorganisms used for producing pyridoxine-5′- ⁇ _darcoside include, for example, bacteria belonging to the genus Leifsonia (Le: Lfsonia) and genus Paenibacillus; actinic radiation belonging to the genus Pseudonocardia.
  • pyridoxin-15 (Sometimes referred to as "1- ⁇ -dalcoside-selective production bacterium”) can be easily selected by those skilled in the art by the method specifically described in Examples of the present invention.
  • pyridoxine-15,1a-darcoside-selective producing bacteria include, for example, Le ⁇ sonia aquatics, Paenibacillus alvei, sydno force 7
  • strains include, for example, Le sonia aquatica IF0 15710, Paenibacillus alvei IFO 3343, Pseudonocardia autotrophics IFO 12743, Cryptococcus albidus IFO 0385, Cryptococcus terreus IFO 0727, Coriolus fibula IFO 4949, Coriolus 917 s , Coriolus unicolor IFO 6265, Coriolus versicolor I AM 13013, Eurotium glabrum JCM 1967, Flammulina velutipes IFO 8329, Ganoderma applanatum IFO 31147, Gliocladiura aureum IFO 9055, Gliocladium virens I AM 5061, Helicostylum nigricans 754, helicostylum nigricans Pithomyces atro-olivaceus IFO 6651, Schizophyllum commune IFO 4928, Schizophyllum commune IFO
  • All of the above microbial strains are available from the Microbial Storage Agency. Culture conditions for the microorganisms are performed by a commonly used method, and are performed in a medium suitable for each of bacteria, fungi, and yeast. Any medium may be used as long as it is a commonly used medium and any medium capable of growing these microorganisms and appropriately containing assimilable carbon sources, nitrogen sources, inorganic substances, and necessary growth promoting substances. Any of a liquid medium and a solid medium may be used, and either a synthetic medium or a natural medium can be used.
  • any carbon compound that can assimilate and grow cells can be used.
  • sugars such as gnorecose, fructose, manoletoose, sucrose, dextrin, soluble starch, gelatinized starch, sorbitol, alcohols such as methanol, ethanol, glycerol, fumaric acid, citric acid, acetic acid, propionic acid, etc.
  • Organic acids and their salts, hydrocarbons such as paraffin, molasses, rapeseed oil and the like can be used alone or in combination.
  • Nitrogen sources include ammonium salts of inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid, ammonium salts of organic acids such as fumaric acid and citric acid, nitrates such as sodium nitrate and potassium nitrate, yeast extract, peptone , Meat extract, corn steep liquor, processed soybeans, and organic nitrogen sources such as urea can be used alone or in combination.
  • the inorganic salts potassium, sodium, calcium, magnesium, manganese, iron, and other sulfates, hydrochlorides, carbonates, nitrates, phosphates, and the like can be used alone or in combination.
  • nutrients used for normal culture such as vitamins, may be added as needed.
  • the culture can be performed under shaking culture, under aeration conditions using a jar armmenter, or under anaerobic conditions.
  • the pH of the medium is preferably in the range of 3 to 1 °
  • the temperature is preferably in the range of 10 to 50 ° C.
  • the culture time is preferably in the range of 10 to 500 hours, but is not limited thereto. It is determined as appropriate for each microorganism.
  • the cultures of the microorganisms or the treated cells obtained by subjecting the cells of the separated microorganisms to appropriate treatment may be used. Good.
  • the treated cells include freeze-dried cells, treated products obtained by treating with toluene, acetone, and the like.Further, the cells are further immobilized by a known immobilization method, for example, an inclusive method, and carrier binding. Immobilized products immobilized by the method, crosslinking method, etc. Wear.
  • the inclusive method is a method using a natural polymer such as carrageenan or alginic acid or a synthetic polymer using a monomer or prepolymer
  • the carrier binding method is a method of adsorbing to chitosan or the like
  • the crosslinking method is dartartaldehyde or the like. The method used is mentioned.
  • a crushed or extracted cell may be used as the processed cell.
  • the crushed cells can be obtained by a known cell crushing method, for example, an ultrasonic crushing method, a French press crushing method, a glass bead crushing method, a dynomill crushing method, and the like.
  • the extract of the cells can be obtained by removing the cells from the cell frame by centrifugation or the like.
  • the cell extract can be immobilized as a crude enzyme solution in the same manner as the cells and used as immobilized enzyme.
  • the crude enzyme solution can be purified and used as a purified enzyme by a combination of salting-out method using ammonium sulfate precipitation, concentration method using an ultrafiltration membrane, separation by ion exchange chromatography, hydrophobic interaction chromatography or gel filtration chromatography. it can.
  • the treated bacterial cell refers to the above-mentioned broken cells of the bacterial cells, ground materials, extracts, immobilized bacterial cells, or crude or purified enzymes isolated therefrom, and immobilization of these enzymes It is used as a concept that includes immobilized immobilized enzymes.
  • the immobilized cells or enzyme By immobilizing a crude or purified enzyme on a carrier as a treated cell, the immobilized cells or enzyme can be used repeatedly, and as a result, pyridoxine-1,5 ⁇ -darcoside is continuously produced in large amounts. It can be produced from pyridoxine or a salt thereof.
  • the target product When used as immobilized cells or immobilized enzymes, the target product can also be produced by a continuous method. By conducting liquid continuously for one year from the month, pyridoxine-1 5'- ⁇ -darcoside can be mass-produced continuously at low cost.
  • the glycosylation reaction can be performed simultaneously with the culture of the microorganism.
  • a sugar donor is contained in the culture medium of the microorganism for the reaction.
  • an ⁇ 1 ⁇ 3 conjugate of glucose such as sucrose, starch, or nigerose
  • an ⁇ 1 ⁇ 2 conjugate of glucose such as kojibiose, or a mixture thereof
  • the starchy substance is an intermolecular darco Any type of starch can be used as long as it can cause silation.
  • soluble starch, gelatinized starch, amylose, amylopectin, maltose, dextrin and the like can be mentioned.
  • Pyridoxine as a substrate may be used in the form of a salt thereof, such as hydrochloric acid salt. Pyridoxine or a salt thereof can be dissolved in a suitable organic solvent and used for the reaction.
  • a suitable organic solvent used here, hexane, ethyl acetate, ether, acetone, ethanol and the like can be used alone or in combination, and further, the solution can be used as an aqueous solution.
  • the concentration of pyridoxine or a salt thereof is not particularly limited, but for example, when pyridosiquin hydrochloride is used, 0.01 to 1 M is preferable.
  • the reaction temperature is preferably 10 to 70 ° C.
  • the pH is preferably 3 to 10, and the reaction is preferably performed for 1 to 500 hours.
  • a buffer such as a phosphate buffer can be used.
  • the target substance is isolated by the ability to crystallize the target substance by filtration such as centrifugation or ultrafiltration, or by removing the cells or treated cells from the reaction solution and performing appropriate post-treatment. can do.
  • a pyridoxine 15,1 ⁇ -glucoside fraction can be collected from the obtained supernatant or filtrate using activated carbon or ion exchange resin chromatography.
  • the ion-exchange resin chromatography is preferably carried out by a simulated moving bed method using a ⁇ a-type or Ca-type cation exchange resin.
  • pyridoxine_5, __ ⁇ -darcoside can be crystallized by a decoction crystallization method, a cooling crystallization method, a crystallization method with ethanol, or the like.
  • the desired product can be purified by crystallization.
  • boric acid In order to increase the selectivity and increase the production of pyridoxine-15,1- ⁇ -darcoside, boric acid must be added to the culture medium when the cells are treated with cells, cultures, or processed cells. And / or borate may be added.
  • borate a salt with an alkali metal such as sodium or potassium is used.
  • an alkali metal such as sodium or potassium
  • sodium metaborate, disodium tetraborate, sodium pentaborate, sodium hexaborate, octaborate Sodium and sodium diborate can also be used as borate buffer, borate When used as a buffer, the pH can be adjusted to around 5 to 8 with boric acid, succinic acid, or the like.
  • the amount of boric acid and Z or borate to be added is not particularly limited, and it is possible to select an appropriate concentration using the 5 ′ selectivity in glycoside as an index. On the other hand, it may be set to about 0.01 to 1M.
  • glycosylation reaction with a known pyridoxine glycosylase.
  • glycosylase include ⁇ -dalcosidase, transdarcosidase, cyclodextrin glucanotransferase (CGTase) and the like.
  • the detection and quantification of pyridoxine_5, - ⁇ _darcoside can be performed, for example, by HPLC, and the purity of the target compound can be quantified by the peak area ratio.
  • PN_ by glycosidation reactions associated with culture 5 scraped lawn 1-2 loopful of generating various microorganisms one a _G, 0. 1% pyridinium Dokishin hydrochloride (PN ⁇ HC 1)
  • Table 1 5 mL Z Tube
  • PN ⁇ HC 1 0. 1% pyridinium Dokishin hydrochloride
  • the obtained supernatant was subjected to HP LC analysis under the conditions shown in Table 2 below, and pyridoxin-15, - ⁇ -glucoside (PN-5'-a-G) or pyridoxine_4 in the culture broth was analyzed. , One ⁇ -darcoside ( ⁇ -4, -a-1G) was quantified, and the 5 ′ selectivity (%) was determined by the following equation.
  • selectivity (%) (PN-5, peak area of G-1 ZPN-4,-peak area of aG + peak area of PN-5) XI 0 0 (Medium composition)
  • Table 3 shows the strains that produced PN-5 ⁇ -G at a production rate of 1.5% or more from PN during UV325nm culture and had a 5-selectivity of 75% or more.
  • NCIMB National Colellection of Industrial and Marine Bacteria, Ltd.
  • Example 2 was used bacteria PN-5, product of _ a _G
  • K-PB potassium phosphate buffer
  • BB borate buffer
  • the 5 'selectivity was increased to 97% or more when the borate buffer was used, as compared with the case where the potassium phosphate buffer was used for the reaction solution.
  • Example 3 Effect of boric acid on PN-5'- ⁇ -G production reaction using bacterial cells Verticillium dahliae JCM 9510, Verticillium dahliae illFO 9765, schizophyllum commune IAM 9006 and the like according to the method of Example 2.
  • the effect of boric acid addition on the production of PN-5,1-a-G by various microorganisms of Coriolus pubescens IFO 9782 was investigated. Table 5 shows the results. In all strains, the 5 'selectivity was higher when borate buffer was used than when potassium phosphate buffer was used as the reaction solution. Table 5
  • K-PB potassium phosphate buffer
  • Example 4 Effect of boric acid on enzymatic PN-5, -G production reaction
  • Hi-Darcosidase was used as an enzyme in rice
  • G9259 from rice manufactured by Sigma
  • trans-dalcosidase was used as trans-glucosidase from Aspergillus niger manufactured by Amano Pharmaceutical.
  • L Amano and CGT ase are from Bacillus macerans Anchisym was used.
  • Pyridoxine hydrochloride (PN.HC1) was used by Daiichi Fine Chemical Co., Ltd.
  • dextrin was used by Matsutani Chemical Industry Co., Ltd. TK-16.
  • the HPLC analysis was performed under the conditions described in Example 1.
  • reaction solution having the composition shown in Table 6 below was placed in a glass sample tube, protected from light with aluminum foil, incubated in a shaking thermostat at 30 ° C, and reacted.
  • the acetate buffer was prepared by adding sodium hydroxide to sodium acetate buffer to adjust ⁇ H to 7.0, and the borate buffer was adjusted to 7.0 with boric acid.
  • Table 7 shows the results of the reaction.
  • Table 6 a Glucosidase (SIGMA) 20U / mL
  • PN-5'-aG 0 1.72 3.06
  • the ratio of the PN-4,1-a-G to PN-5,1- ⁇ -G of the product was 29:71 9:91, the production ratio of ⁇ ⁇ —5, one ⁇ —G was clearly improved.
  • PN-oligo is a glycoside in which two or more glucose molecules are ⁇ -linked to pyridoxine.
  • PN-Oligo 0 0 0 PN-4, - ⁇ -G after 144 hours of reaction for CGTase: PN-5 one ⁇ -G is 38:62 when an acetate buffer equivalent to ⁇ (0.15M) is added.
  • 1 712 equivalents of ⁇ (0.075 ⁇ ) borate buffer was added, the ratio was 29:71.
  • ⁇ equivalent (0.15M) of borate buffer was added, 17 17. : 83.
  • ⁇ ⁇ ⁇ ⁇ _4, - ⁇ -G: PN-5,1H-1G after 70 hours of reaction with transdarcosidase was 53:47 when an acetate buffer equivalent to PN (0.15M) was added.
  • pyridoxine-5′-dalcoside can be easily and efficiently produced from pyridoxine or a salt thereof.

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Abstract

A process for producing pyridoxine-5’-α-glucoside by glucosylating pyridoxine at the 5’-position which involves the step of treating pyridoxine or its salt with microbial cells capable of selectively glucosylating pyridoxine at the 5’-position to thereby form pyridoxine-5’-α-glucoside, a culture thereof or processed cells thereof; and a process for producing pyridoxine-5’-α-glucoside by treating pyridoxine or its salt with a glucosylating enzyme, characterized in that the treatment is carried out in the presence of boric acid and/or a boric acid salt to thereby enhance the selectivity of the glucosylation at the 5’-position. According to these methods, pyridoxine-5’-α-glucoside can be easily and effectively produced from pyridoxine or its salt.

Description

明 細 書 ピリ ドキシン一 5， —ひ—ダルコシドの製造方法 技術分野  SPECIFICATION PROCESS FOR PRODUCING PYRIDOXIN-1,5-HI-DALCOSIDE
本発明は、 ピリ ドキシン一 5' —α_ダルコシドの製造方法、 より詳しくは、 ピリ ドキシンの 5， 位を選択的に配糖化してピリ ドキシン一 5， 一 α—ダルコシ ドを生成する能力を有する微生物菌株を用いるピリ ドキシン— 5， 一 α_ダルコ シドの製造方法に関する。 The present invention relates to a method for producing pyridoxine-1 5′-α_darcoside, and more particularly, to the ability to selectively glycosylate pyridoxine at the 5-position to produce pyridoxine-15,1-α-darcoside. pyridinium Dokishin using a microorganism strain having - 5, a method for producing one alpha _ Darco Sid.
本発明はまた、 ピリ ドキシン又はその塩を配糖化酵素で処理してピリ ドキシン 一 α—ダルコシドを製造する方法において、 5， 位の配糖化の選択性を高める方 法に関する。 背景技術 The present invention also relates to a method for producing pyridoxine mono- α -darcoside by treating pyridoxine or a salt thereof with a glycosidase, the method of enhancing selectivity for glycosylation at the 5-position. Background art
ピリ ドキシン （ΡΝ) は、 ピリ ドキサール （PL)、 ピリ ドキサミン （PM) と ともにビタミン B 6作用を持つ物質の一つである。 細胞に取り込まれた PNはリ ン酸化されてピリ ドキシン一 5， 一リン酸 （PNP) となり、 さらに酸化されて ピリ ドキサール一 5' —リン酸 （PLP) に変換され、 アミノ酸代謝にあずかる 酵素の補酵素として重要な役割を果たしている。  Pyridoxine (ΡΝ) is one of the substances that has vitamin B6 action together with pyridoxal (PL) and pyridoxamine (PM). PN taken into cells is phosphorylated to form pyridoxine-15, monophosphate (PNP), which is further oxidized to pyridoxal-5'-phosphate (PLP), an enzyme that participates in amino acid metabolism. It plays an important role as a coenzyme.
ピリ ドキシンの塩酸塩 （PN . HC 1) は、 水によく溶けるが、 光に対して不 安定であり、 また、 酸味や苦味があるという問題を有している。 一方、 その配糖 体であるピリ ドキシン一 α—ダルコシドは、光安定性に優れており [J. Vitaminol.， 17, 121-124(1971)]、 酸味や苦味がないかあるいは緩和されており、 また経口投 与された場合、 腸管でそのままの形で吸収され、 速やかにピリ ドキサールリン酸 に変換されることが動物実験で確認されており、 医薬としての有用性が高い  Pyridoxine hydrochloride (PN.HC1) is soluble in water but is unstable to light, and has problems of acidity and bitterness. On the other hand, its glycoside, pyridoxine-α-darcoside, has excellent photostability [J. Vitaminol., 17, 121-124 (1971)], and has no or less acidity or bitterness. Also, when administered orally, it has been confirmed in animal experiments that it is absorbed as it is in the intestinal tract and is rapidly converted to pyridoxal phosphate, which is highly useful as a pharmaceutical.
[J.Nutr. Sci.Vitaminol. , 42, 377-386 (1996)]。 ピリ ドキシン一 α _ダルコシド には 5， 位と 4' 位の配糖体が存在するが、 ピリ ドキシン一 5， 一 α—ダルコシ ドはホモジェネートされた肝細胞によって速やかに ΡΝに加水分解されることか ら、 医薬品や健康食品として有用性が高く、 大量生産が望まれるところである。 これまで報告されている微生物菌体又はそれより生成した酵素を用いたピリ ド キシン一 5， _ α—ダルコシドの製造法としては、 Micrococcus属菌体を用いる 方法 [特公昭 46-33198 (1971)、 J. Vitaminology 15， 142-150 (1969)、 [J. Nutr. Sci. Vitaminol., 42, 377-386 (1996)]. Pyridoxine-1 α-darcoside has glycosides at positions 5 and 4 ', but pyridoxine-1-5,1-α-darcoside is rapidly hydrolyzed to ΡΝ by homogenized hepatocytes. Or Therefore, they are highly useful as pharmaceuticals and health foods, and mass production is desired. As a method for producing pyridoxine-15, _α-darcoside using a microorganism cell or an enzyme produced therefrom, a method using a microorganism of the genus Micrococcus [Japanese Patent Publication No. 46-33198 (1971)] , J. Vitaminology 15, 142-150 (1969),
J. Vitaminology 15, 160 - 166 (1969)、特公昭 49-18230 (19<74)、及ぴ J. Vitaminology 15， 167-173 (1969) ]、 Mucor javanicus IF04570から精製した酵素を用いる方法 [応用糖質科学 43, 369-372 (1996)、 Methods in Enzymology, 280， 66-71 (1997) ]、 Bacillus macerans又は Bacillus stearothermophilus由来の CGTase (シクロテ キストリングルカノトランスフェラーゼ） を用いる反応 [Methods in Enzymology, 280, 61-71 (1997) ]などがあるが、 いずれも生成率が低かったり、 5 '選択率が低 く、生産量において満足のいく結果が得られていない。 また、 5 '選択率が低い場 合は、 副生成物のピリ ドキシン一 4， _ α _ダルコシドの量が多いため、 これを 除去するためにさらなる精製処理が必要で工程が複雑になり、 また、 ピリ ドキシ ンー 4 ' — α _ダルコシドの基質としての再利用が難しくなるなどの問題があつ た。 発明の開示 J. Vitaminology 15, 160 - 166 ( 196 9), JP-B 4 9 -18 230 (1 9 <74),及Pi J. Vitaminology 15, 167-173 (1969) ], using enzymes purified from Mucor javanicus IF04570 Reaction [Applied Glycoscience 43, 369-372 (1996), Methods in Enzymology, 280, 66-71 (1997)], Reaction using CGTase (cyclotexturlucanotransferase) derived from Bacillus macerans or Bacillus stearothermophilus [Methods in Enzymology, 280, 61-71 (1997)], but none of them has a low production rate or low 5 'selectivity, and satisfactory results have not been obtained in terms of production volume. In addition, when the 5 'selectivity is low, the amount of the by-product pyridoxine-14, _α_darcoside is large, so that a further purification treatment is required to remove it, and the process becomes complicated. However, there were problems such as difficulty in reusing pyridoxin-4'-α_darcoside as a substrate. Disclosure of the invention
従って、 本発明の課題は、 ピリ ドキシンの 5 ' 位を選択的に配糖化する方法を 提供することにある。 より具体的には、 ピリ ドキシン一 5， 一 α—ダルコシドを 生成する能力を有する微生物菌株を見出し、 当該微生物の菌体、 その培養物、 又 は菌体処理物を用いてピリ ドキシン又はその塩からピリ ドキシン一 5， 一 α—グ ルコシドのみを容易にかつ効率よく製造する方法を提供することにある。  Therefore, an object of the present invention is to provide a method for selectively glycosylating the 5′-position of pyridoxine. More specifically, a microbial strain capable of producing pyridoxine-15,1-α-darcoside was found, and pyridoxine or a salt thereof was obtained by using the cells, cultures, or processed cells of the microorganism. An object of the present invention is to provide a method for easily and efficiently producing only pyridoxine 15, 1 α-glucoside from glycerol.
本発明のもうひとつの課題は、 ピリ ドキシン又はその塩を配糖化酵素で処理し てピリ ドキシン一ひ _ダルコシドを製造する方法において、 5， 位の配糖化の選 択性を高める方法を提供することにある。  Another object of the present invention is to provide a method for producing pyridoxine mono-Dalcoside by treating pyridoxine or a salt thereof with a glycosylase, which enhances the selectivity of glycosylation at the 5-position. It is in.
本努明者らは、 上記課題を解決すベく属種が同定されている微生物を対象とし て 5， 選択性が高い菌株を広くスクリーニングした結果、 細菌、 真菌の中に 5， 選択率が特に高い微生物を見出した。 さらに、 ピリ ドキシン又はその塩を前記微 生物の菌体、 その培養物、 菌体処理物、 または配糖化酵素で処理してピリ ドキシ ン—ひ—ダルコシドを製造する際に、 ホウ酸及ぴ 又はホウ酸塩の存在下で処理 を行うと 5， 位の配糖化の選択性が高まることを見出した。 本発明はこれらの知 見に基にして完成されたものである。 The present researchers have screened a wide variety of highly selective strains for microorganisms whose genus species have been identified to solve the above-mentioned problems. As a result, the selectivity among bacteria and fungi was 5, Especially high microorganisms were found. Further, pyridoxine or a salt thereof is When producing pyridoxine-hydarcoside by treating with a bacterial cell, a culture thereof, a treated bacterial cell, or a glycosidase, the treatment is performed in the presence of boric acid and / or borate And that the selectivity for glycosylation at the 5th position is enhanced. The present invention has been completed based on these findings.
即ち、 本発明は、 ピリドキシンの 5 ' 位を配糖化してピリ ドキシン一 5， - a —ダルコシドを製造する方法であって、 下記のいずれかの属に属し、 ピリ ドキシ ンの 5 ' 位を選択的に配糖化してピリ ドキシン一 5， - a -ダルコシドを生成す る能力を有する微生物の菌体、 その培養物、 又は菌体処理物でピリ ドキシン又は その塩を処理する工程を含む方法を提供するものである。  That is, the present invention relates to a method for producing pyridoxine-5, -a-darcoside by glycosylation of the 5′-position of pyridoxine, which belongs to any one of the following genera. A method comprising the step of treating pyridoxine or a salt thereof with a microorganism, a culture thereof, or a treated product of a microorganism capable of selectively glycosylating and producing pyridoxine-1,5-a-darcoside. Is provided.
レイフソニァ （Leifsonia)属 Genus Leifsonia
パェニノ チノレス（Paenibacillus)属 Paenibacillus genus
シユードノカルアイァ (Pseudonocardia) Jh Pseudonocardia Jh
クリプ卜コッカス (Cryptococcus)属 Genus Cryptococcus
コリオルス （Coriolus)属 Coriolus genus
ユーロチウム (Eurotium)属 Genus Eurotium
フラムリナ (Flammul ina)属 Genus Flammul ina
ガノデルマ (Ganoderma)属 Ganoderma
グリオクラディウム （Gliocladium)属 Gliocladium genus
ヘリコステイノレム (Helicostylum)属 Helicostylum
モノレティエレラ (Mortierella)属 Genus Mortierella
ピソマイセス （Pithomyces)属  Genus Pithomyces
シゾフィルム (Schizophyllum)属 Genus Schizophyllum
トラメテス （Trametes)属  Genus Trametes
ベノレティシリウム (Verticillium) 属 Genus Verticillium
上記発明の好ましい態様によれば、 ホウ酸及び/又はホウ酸塩の存在下で処理 を行う上記方法が提供される。  According to a preferred embodiment of the present invention, there is provided the above method, wherein the treatment is performed in the presence of boric acid and / or borate.
また、 別の観点からは、 ピリ ドキシン又はその塩を配糖ィヒ酵素で処理してピリ ドキシン一ひ—ダルコシドを製造する方法において、 ホウ酸及び/又はホウ酸塩 の存在下で処理を行い 5， 位の配糖化の選択性を高める方法、 及びピリ ドキシン 又はその塩を配糖化酵素で処理してピリ ドキシン一 α—ダルコシドを製造する方 法において 5 ' 位の配糖化の選択性を高めるための試薬であって、 ホウ酸及び Ζ 又はホウ酸塩を含む試薬が本発明により提供される。 発明を実施するための最良の形態 In another aspect, the present invention provides a method for producing pyridoxine mono-darcoside by treating pyridoxine or a salt thereof with a glycoside enzyme, wherein boric acid and / or borate is used. In the method of treating pyridoxine or a salt thereof with glycosidase to produce pyridoxine mono-α-darcoside, the method of treating pyridoxine or its salt with glycosidase is carried out in the presence of The present invention provides a reagent for enhancing the glycosylation selectivity, which reagent comprises boric acid and 又 は or borate. BEST MODE FOR CARRYING OUT THE INVENTION
本発明の方法は、 ピリ ドキシン一 5 ， 一 一ダルコシドを製造するにあたり、 ピリ ドキシン又はその塩を原料化合物として用い、 ピリ ドキシンの 5， 位を選択 的に配糖ィヒしてピリ ドキシン一 5 ' — α—ダルコシドを生成する能力を有する微 生物の菌体、 その培養物、 又は菌体処理物を用いることを特徴としている。  In the method of the present invention, pyridoxine or a dalcoside is produced by using pyridoxine or a salt thereof as a starting compound and selectively glycosylating the 5-position of pyridoxine to form pyridoxine. '— It is characterized by using bacterial cells of microorganisms capable of producing α-darcoside, cultures thereof, or processed cells.
本発明においてピリ ドキシン— 5 ' — α _ダルコシドの生産に使用する微生物 は、 例えばレイフソニァ （Le:Lfsonia)属、パェニバチルス（Paenibacillus)属に属 する細菌；シユードノカルディア（Pseudonocardia)属に属する放線菌； クリプト コッカス （Cryptococcus) 属に属する酵母； コリオノレス （Coriolus) 属、 ユーロ チウム (Eurotium) 属、 フラムリナ (Flammulina) 属、 ガノデルマ (Ganoderma) 属、 グリオクラディウム （Gliocladium) 属、 ヘリコステイルム （Helicostylum) 属、 モルティエレラ （Mortierella) 属、 ピソマイセス （Pithomyces) 属、 シゾフ イノレム (Schizophyllum) 属、 卜ラメテス (Trametes) 属、 ベノレティシジゥム In the present invention, microorganisms used for producing pyridoxine-5′-α_darcoside include, for example, bacteria belonging to the genus Leifsonia (Le: Lfsonia) and genus Paenibacillus; actinic radiation belonging to the genus Pseudonocardia. Bacteria; yeast belonging to the genus Cryptococcus; genus Coriolus, genus Eurotium, genus Flammulina, genus Ganoderma, genus Gliocladium, helicopterium Helicostylum), Mortierella, Pithomyces, Schizophyllum, Trametes, Benoletiszyme
(Vert ici Ilium) に属するカビから選択される。 上記の属に属する微生物であつ て、 ピリ ドキシンの 5， 位を選択的に配糖化し、 ピリ ドキシン一 5 ， _ α _ダル コシドを生成する能力を有する菌株（以下、 「ピリ ドキシン一 5 ， 一 α—ダルコシ ド選択的生産菌」 と呼ぶ場合がある） は、 本発明の実施例に具体的に説明した方 法により当業者が容易に選択可能である。 (Vert ici Ilium). A microorganism belonging to the above genus, which is capable of selectively glycosylating the 5-position of pyridoxine to produce pyridoxine-15, _α_darcoside (hereinafter, “pyridoxin-15, (Sometimes referred to as "1- α -dalcoside-selective production bacterium") can be easily selected by those skilled in the art by the method specifically described in Examples of the present invention.
ピリ ドキシン一 5 ，一 a—ダルコシド選択的生産菌の具体例としては、例えば、 レイフソニァ アクアティカ （Leぱ sonia aquatics)、 パェニバチルス アルべィ (Paenibacillus alvei) , シユードノ力 7レディァ アウトトロブイ力  Specific examples of the pyridoxine-15,1a-darcoside-selective producing bacteria include, for example, Le ぱ sonia aquatics, Paenibacillus alvei, sydno force 7
(Pseudonocardia autotrophica クリプトコッカス ァノレビグス (Cryptococcus albidus)、クジプ卜 クカス テレクス (Cryptococcus terreus)、 V才ノレス フ イブラ (Coriolus fibula)、 コジオノレス ヒノレステュス (Coriolus hirsutus) ^ コリオノレス プべセンス (Coriolus pubescens)、 コリオノレス ュニカラー(Pseudonocardia autotrophica Cryptococcus albidus), Cryptococcus terreus, V-year-old Ibra (Coriolus fibula), Kozionores hinorestus (Coriolus hirsutus) ^ Corionolus pubescens, Corionores unicolor
(Coriolus unicolor) コリオルス ベルシカフー (Coriolus versicolor) N ュ 一口チウム グラブラム (Eurotium glabrum)、 フラムリナ ベノレティぺス (Coriolus unicolor) Koriorusu Berushikafu (Coriolus versicolor) N Interview bite lithium Guraburamu (Eurotium glabrum), Furamurina Benoretipesu
(Flaramulina velutipes)、ガノテノレマ ァプフナァュム (Ganoderma applanatum)、 グリオクラディウム ァウレゥム （Gliocladium aureum)、 グリオクラディウム ビレンス（Gliocladium virens)、ヘリコステイノレム ニグリカンス（Helicostylum nigricans)、 モルティレラ アルピナ （Mortierella alpina)、 ピソマイセス ァ 卜口一オリバセウス (Pithomyces atro-olivaceus) ^ シゾフィルム コミュネ (Flaramulina velutipes), Ganoderma applanatum (Ganoderma applanatum), Gliocladium aureum, Gliocladium virens (Gliocladium virens), Helicostinella nigersans (Helicosty lamina pirina) Pithomyces atro-olivaceus ^ Shizofilm commune
(Schizophyllum commune; ^卜フメテス オリエンタジス (Trametes orientalis)、 ベノレテイシリゥム ァノレポーアトノレム (Verticillium albo-atrum)、 ベノレティシ リウム ダーリアェ （Verticillium dahliae)、 ベルテイシリゥム トリコノレプス(Schizophyllum commune; ^ Trametes orientalis), Benoretheisirimu anorepoatonorem (Verticillium albo-atrum), Verenoillium dahliae, Verticillium dahliae,
(Verticillium tricorpus) に属する微生物の菌株を挙げることができる。 その 代表的な菌株としては、 例えば、 Le sonia aquatica IF0 15710、 Paenibacillus alvei IFO 3343、 Pseudonocardia autotrophics IFO 12743、 Cryptococcus albidus IFO 0385、 Cryptococcus terreus IFO 0727、 Coriolus fibula IFO 4949、 Coriolus hirsutus IFO 4917、 Coriolus pubescens IFO 9782、 Coriolus unicolor IFO 6265、 Coriolus versicolor I AM 13013、 Eurotium glabrum JCM 1967、 Flammulina velutipes IFO 8329、 Ganoderma applanatum IFO 31147、 Gliocladiura aureum IFO 9055、 Gliocladium virens I AM 5061、 Helicostylum nigricans IFO 8091、 Mortierella alpina CBS 754. 68、 Pithomyces atro-olivaceus IFO 6651、 Schizophyllum commune IFO 4928、 Schizophyllum commune IFO 4929、 Schizophyllum commune IFO 6502、 Schizophyllum commune I AM 9006、 Schizophy丄丄 um commune I AM 13042、 Trametes orientalis IFO 6483、 Verticillium albo-atrum IFO 9435、 Verticillium dahliae IFO 9765、 Verticillium dahliae IFO 31024、 Verticillium dahliae JCM 9509、 Verticillium dahliae JCM 9510、 Verticillium tricorpus IFO 31025が挙げられるが、 これらに限定されることはない。 上記微生物菌株はいず れも微生物保存機関から入手可能である。 前記微生物の培養条件は、 通常用いられる方法で行われ、 細菌、 真菌、 酵母そ れぞれ適した培地で行われる。培地としては、通常使用されるものであればよく、 これら微生物が生育することができ、 かつ資化可能な炭素源、 窒素源、 無機物及 び必要な生育促進物質を適当に含有する培地であれば液体、 固体のいずれでも良 く、 また合成培地、 天然培地のいずれも用いることができる。 (Verticillium tricorpus). Representative strains include, for example, Le sonia aquatica IF0 15710, Paenibacillus alvei IFO 3343, Pseudonocardia autotrophics IFO 12743, Cryptococcus albidus IFO 0385, Cryptococcus terreus IFO 0727, Coriolus fibula IFO 4949, Coriolus 917 s , Coriolus unicolor IFO 6265, Coriolus versicolor I AM 13013, Eurotium glabrum JCM 1967, Flammulina velutipes IFO 8329, Ganoderma applanatum IFO 31147, Gliocladiura aureum IFO 9055, Gliocladium virens I AM 5061, Helicostylum nigricans 754, helicostylum nigricans Pithomyces atro-olivaceus IFO 6651, Schizophyllum commune IFO 4928, Schizophyllum commune IFO 4929, Schizophyllum commune IFO 6502, Schizophyllum commune I AM 9006, Schizophy 丄 丄 um commune I AM 13042, Trametes orientalis IFO 6483, Verticillium 9435, Verticillium albo-atrum dahliae IFO 9765, Verticillium dahliae IFO 31024, Verticillium dahliae JCM 9509, Verticilli um dahliae JCM 9510, Verticillium tricorpus IFO 31025, but is not limited thereto. All of the above microbial strains are available from the Microbial Storage Agency. Culture conditions for the microorganisms are performed by a commonly used method, and are performed in a medium suitable for each of bacteria, fungi, and yeast. Any medium may be used as long as it is a commonly used medium and any medium capable of growing these microorganisms and appropriately containing assimilable carbon sources, nitrogen sources, inorganic substances, and necessary growth promoting substances. Any of a liquid medium and a solid medium may be used, and either a synthetic medium or a natural medium can be used.
炭素源としては、 菌体が資化し生育できる炭素化合物であればいずれでも使用 可能である。 例えば、 グノレコース、 フルクトース、 マノレトース、 スクロース、 デ キストリン、 可溶性デンプン、 糊化デンプン、 ソルビトールなどの糖類、 メタノ ール、 エタノール、 グリセロールなどのアルコール類、 フマル酸、 クェン酸、 酢 酸、 プロピオン酸などの有機酸類及びその塩類、 パラフィンなどの炭化水素類、 糖蜜、 菜種油などを単独で又は混合して使用することができる。  As a carbon source, any carbon compound that can assimilate and grow cells can be used. For example, sugars such as gnorecose, fructose, manoletoose, sucrose, dextrin, soluble starch, gelatinized starch, sorbitol, alcohols such as methanol, ethanol, glycerol, fumaric acid, citric acid, acetic acid, propionic acid, etc. Organic acids and their salts, hydrocarbons such as paraffin, molasses, rapeseed oil and the like can be used alone or in combination.
• 窒素源としては、 塩酸、 硫酸、 硝酸、 リン酸などの無機酸のアンモニゥム塩、 フマル酸、 クェン酸などの有機酸のアンモニゥム塩、 硝酸ナトリウム、 硝酸カリ ゥムなどの硝酸塩、酵母エキス、ペプトン、肉エキス、 コーンスティープリカ一、 大豆加工品、 尿素などの有機窒素源を単独で又は混合して用いることができる。 無機塩類としては、 カリウム、 ナトリウム、 カルシウム、 マグネシウム、 マン ガン、 鉄などの硫酸塩、 塩酸塩、 炭酸塩、 硝酸塩、 リン酸塩などをそれぞれ単独 で又は混合して用いることができる。 さらに、 ビタミン類などの通常の培養に用 いられる栄養源を適宜添カ卩してもよレ、。 • Nitrogen sources include ammonium salts of inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid and phosphoric acid, ammonium salts of organic acids such as fumaric acid and citric acid, nitrates such as sodium nitrate and potassium nitrate, yeast extract, peptone , Meat extract, corn steep liquor, processed soybeans, and organic nitrogen sources such as urea can be used alone or in combination. As the inorganic salts, potassium, sodium, calcium, magnesium, manganese, iron, and other sulfates, hydrochlorides, carbonates, nitrates, phosphates, and the like can be used alone or in combination. Furthermore, nutrients used for normal culture, such as vitamins, may be added as needed.
培養は、 振とう培養、 ジャーフアーメンターなどを用いた通気条件下で行うほ 力、嫌気的条件下で行うことができる。培地の p Hは 3〜 1◦の範囲が好ましく、 温度は 1 0〜 5 0 °Cの範囲が好ましく、培養時間は 1 0〜 5 0 0時間が好ましい が、 これらの限定されることなく、 それぞれの微生物によって適宜決められる。 本発明の方法では、培養物から分離した微生物の菌体のほか、微生物の培養物、 あるいは分離した微生物の菌体を適宜の処理に付して得られる菌体処理物を用レ、 てもよい。 菌体処理物としては、 例えば、 菌体の凍結乾燥物、 トルエン、 ァセト ンなどで処理して得られる処理物のほか、 さらに、 菌体を公知の固定化法、 例え ば包括法、 担体結合法、 架橋法などで固定化した固定化物などを挙げることがで きる。 包括法としては、 カラギーナンやアルギン酸などの天然高分子又はモノマ 一やプレボリマーによる合成高分子を用いる方法、 担体結合法としては、 キトサ ンなどに吸着させる方法、 架橋法としては、 ダルタルアルデヒドなどを用いる方 法が挙げられる。 The culture can be performed under shaking culture, under aeration conditions using a jar armmenter, or under anaerobic conditions. The pH of the medium is preferably in the range of 3 to 1 °, the temperature is preferably in the range of 10 to 50 ° C., and the culture time is preferably in the range of 10 to 500 hours, but is not limited thereto. It is determined as appropriate for each microorganism. In the method of the present invention, in addition to the cells of the microorganisms isolated from the culture, the cultures of the microorganisms or the treated cells obtained by subjecting the cells of the separated microorganisms to appropriate treatment may be used. Good. Examples of the treated cells include freeze-dried cells, treated products obtained by treating with toluene, acetone, and the like.Further, the cells are further immobilized by a known immobilization method, for example, an inclusive method, and carrier binding. Immobilized products immobilized by the method, crosslinking method, etc. Wear. The inclusive method is a method using a natural polymer such as carrageenan or alginic acid or a synthetic polymer using a monomer or prepolymer, the carrier binding method is a method of adsorbing to chitosan or the like, and the crosslinking method is dartartaldehyde or the like. The method used is mentioned.
また、 菌体処理物として菌体の破碎物又は抽出物を用いてもよい。 菌体の破碎 物は、 公知の菌体破碎法、 例えば超音波破碎法、 フレンチプレス破碑法、 ガラス ビーズ破碎法、 ダイノミル破枠法などにより得ることができる。 また、 菌体の抽 出物は、前記菌体破枠物から遠心分離などにより菌体を除いて得ることができる。 菌体の抽出物は、 粗酵素液として菌体と同様に固定化して固定ィ匕酵素として用い ることができる。粗酵素液は、硫安沈殿による塩析法、限外濾過膜による濃縮法、 イオン交換クロマトグラフィー、 疎水相互作用クロマトグラフィー又はゲル濾過 クロマトグラフィーによる分離などの組み合わせにより精製して精製酵素として 用いることもできる。 本明細書において、 菌体処理物とは、 上記のような菌体の 破枠物、 磨砕物、 抽出物、 固定化菌体、 又はそれらから単離した粗製もしくは精 製酵素、 それら酵素を固定化した固定化酵素などを含む概念として用いる。  In addition, a crushed or extracted cell may be used as the processed cell. The crushed cells can be obtained by a known cell crushing method, for example, an ultrasonic crushing method, a French press crushing method, a glass bead crushing method, a dynomill crushing method, and the like. The extract of the cells can be obtained by removing the cells from the cell frame by centrifugation or the like. The cell extract can be immobilized as a crude enzyme solution in the same manner as the cells and used as immobilized enzyme. The crude enzyme solution can be purified and used as a purified enzyme by a combination of salting-out method using ammonium sulfate precipitation, concentration method using an ultrafiltration membrane, separation by ion exchange chromatography, hydrophobic interaction chromatography or gel filtration chromatography. it can. In the present specification, the treated bacterial cell refers to the above-mentioned broken cells of the bacterial cells, ground materials, extracts, immobilized bacterial cells, or crude or purified enzymes isolated therefrom, and immobilization of these enzymes It is used as a concept that includes immobilized immobilized enzymes.
菌体処理物として粗製もしくは精製酵素を担体に固定化することにより、 その 固定化菌体又は固定化酵素を繰り返し使用でき、 その結果、 連続的に大量にピリ ドキシン一 5， 一 α—ダルコシドをピリ ドキシン又はその塩から製造することが できる。 固定化菌体又は固定化酵素として使用する場合、 連続法によって目的物 の製造を行うこともでき、 例えばそれらをカラムに充填してバイオリアクターと して、 回分式と同様の反応条件で一力月から一年間連続通液を行うことにより、 ピリ ドキシン一 5 ' — α—ダルコシドを安価に連続的に大量生産することができ る。  By immobilizing a crude or purified enzyme on a carrier as a treated cell, the immobilized cells or enzyme can be used repeatedly, and as a result, pyridoxine-1,5α-darcoside is continuously produced in large amounts. It can be produced from pyridoxine or a salt thereof. When used as immobilized cells or immobilized enzymes, the target product can also be produced by a continuous method. By conducting liquid continuously for one year from the month, pyridoxine-1 5'-α-darcoside can be mass-produced continuously at low cost.
配糖化反応は、 微生物の培養と同時に行わせることも可能である。 その場合、 微生物培養の培地中に糖供与体を含有させ反応させる。 配糖化反応における糖の 供与体としては、 スクロース、 デンプン質、 ニゲロースなどのグルコースの α 1 →3結合体、 コージビオースなどのグルコースの α 1→2結合体、 あるいはこれ らの混合物などが使用される。 上記デンプン質とは菌体が作用して分子間ダルコ シル化を起こしうるデンプン質であればその種類は問わない。 例えば、 可溶性デ ンプン、 糊化デンプン、 アミロース、 アミロぺクチン、 マルトース、 デキス トリ ンなどが挙げられる。 基質としてのピリ ドキシンはその塩を用いてもよく、 塩酸 塩などが挙げられる。 ピリ ドキシン又はその塩は、 適当な有機溶媒に溶解し反応 に供することができる。ここで用いる有機溶媒としては、へキサン、酢酸ェチル、 エーテル、 アセトン、 ェタノールなどを単独で又は組み合わせて、 さらには該溶 解液を水性溶液として用いることができる。 The glycosylation reaction can be performed simultaneously with the culture of the microorganism. In this case, a sugar donor is contained in the culture medium of the microorganism for the reaction. As the sugar donor in the glycosylation reaction, an α1 → 3 conjugate of glucose such as sucrose, starch, or nigerose, an α1 → 2 conjugate of glucose such as kojibiose, or a mixture thereof is used. . The starchy substance is an intermolecular darco Any type of starch can be used as long as it can cause silation. For example, soluble starch, gelatinized starch, amylose, amylopectin, maltose, dextrin and the like can be mentioned. Pyridoxine as a substrate may be used in the form of a salt thereof, such as hydrochloric acid salt. Pyridoxine or a salt thereof can be dissolved in a suitable organic solvent and used for the reaction. As the organic solvent used here, hexane, ethyl acetate, ether, acetone, ethanol and the like can be used alone or in combination, and further, the solution can be used as an aqueous solution.
ピリ ドキシン又はその塩の濃度は特に限定されないが、 例えばピリ ドシキン塩 酸塩を用いる場合、 0 . 0 1〜1 Mが好ましい。また、反応温度は 1 0〜7 0 °C、 p Hは 3〜1 0が好ましく、 1〜5 0 0時間反応させるのが好ましい。 p Hを一 定に維持するためには、 例えば、 リン酸緩衝液などの緩衝液を使用することがで きる。  The concentration of pyridoxine or a salt thereof is not particularly limited, but for example, when pyridosiquin hydrochloride is used, 0.01 to 1 M is preferable. Further, the reaction temperature is preferably 10 to 70 ° C., and the pH is preferably 3 to 10, and the reaction is preferably performed for 1 to 500 hours. To maintain the pH constant, for example, a buffer such as a phosphate buffer can be used.
反応終了後、 遠心分離又は限外濾過などの濾過分離により目的物を晶析させる 力 あるいは反応液から菌体又は菌体処理物を除去して適宜の後処理を施すこと により目的物を単離することができる。 例えば、 得られた上清又は濾液より、 活 性炭やイオン交換樹脂クロマトグラフィーを用いてピリ ドキシン一 5， 一 α—グ ルコシド画分を集めることができる。 イオン交換樹脂クロマトグラフィーは、 Ν a型又は C a型の陽イオン交換樹脂を用いて擬似移動床法によって行うのが好ま しい。 次いで、 得られた画分を濃縮して、 煎糖晶析法、 冷却晶析法、 エタノール による晶析法などによりピリ ドキシン _ 5， _ α—ダルコシドを晶出させること ができ、 必要により再結晶を行って目的物を精製することができる。  After the completion of the reaction, the target substance is isolated by the ability to crystallize the target substance by filtration such as centrifugation or ultrafiltration, or by removing the cells or treated cells from the reaction solution and performing appropriate post-treatment. can do. For example, a pyridoxine 15,1α-glucoside fraction can be collected from the obtained supernatant or filtrate using activated carbon or ion exchange resin chromatography. The ion-exchange resin chromatography is preferably carried out by a simulated moving bed method using a Νa-type or Ca-type cation exchange resin. Subsequently, the obtained fraction is concentrated, and pyridoxine_5, __ α-darcoside can be crystallized by a decoction crystallization method, a cooling crystallization method, a crystallization method with ethanol, or the like. The desired product can be purified by crystallization.
また、 5， 選択率を上げてピリ ドキシン一 5， 一α—ダルコシドの生産量を增 大させるには、 菌体、 培養物、 又は菌体処理物での処理を行うにあたり、 培地に ホウ酸及び/又はホウ酸塩を添加するとよい。 In order to increase the selectivity and increase the production of pyridoxine-15,1- α -darcoside, boric acid must be added to the culture medium when the cells are treated with cells, cultures, or processed cells. And / or borate may be added.
ここで、 ホウ酸塩としては、 ナトリウムやカリウムなどのアルカリ金属との塩 などが用いられ、 例えば、 メタホウ酸ナトリウム、 四ホウ酸ニナトリウム、 五ホ ゥ酸ナトリウム、 六ホウ酸ナトリウム、 八ホウ酸ナトリウム、 二ホウ酸ナトリウ ムなどが挙げられる。 また、 ホウ酸塩緩衝液として用いることもでき、 ホウ酸塩 緩衝液として用いる場合、 ホウ酸、 コハク酸などにより pH5〜8付近に調整し て用いることができる。 Here, as the borate, a salt with an alkali metal such as sodium or potassium is used. For example, sodium metaborate, disodium tetraborate, sodium pentaborate, sodium hexaborate, octaborate Sodium and sodium diborate. It can also be used as borate buffer, borate When used as a buffer, the pH can be adjusted to around 5 to 8 with boric acid, succinic acid, or the like.
ホウ酸及び Z又はホウ酸塩の添加量は特に限定されず、 配糖ィヒにおける 5 ' 選 択性を指標として適宜の濃度を選択することが可能であるが、 例えば、 培地又は 反応液に対し、 0. 0 1〜1M程度とすればよい。  The amount of boric acid and Z or borate to be added is not particularly limited, and it is possible to select an appropriate concentration using the 5 ′ selectivity in glycoside as an index. On the other hand, it may be set to about 0.01 to 1M.
ホウ酸及び 又はホウ酸塩の添カ卩による 5 ' 位配糖化の選択性の向上は、 公知 のピリ ドキシン配糖化酵素による配糖ィ匕反応においても認められる。 配糖化酵素 として、 具体的には α—ダルコシダーゼ、 トランスダルコシダーゼ、 シクロデキ ストリングルカノトランスフェラーゼ （CGT a s e) などが挙げられる。 The improvement in the selectivity of 5'-glycosylation with boric acid and / or borate by addition of syrup is also observed in the glycosylation reaction with a known pyridoxine glycosylase. Specific examples of the glycosylase include α -dalcosidase, transdarcosidase, cyclodextrin glucanotransferase (CGTase) and the like.
なお、 ピリ ドキシン _ 5， — α_ダルコシドの検出及ぴ定量は、 例えば、 HP L Cにより行うことができ、 目的物の純度はピーク面積比により定量することが できる。 実施例  The detection and quantification of pyridoxine_5, -α_darcoside can be performed, for example, by HPLC, and the purity of the target compound can be quantified by the peak area ratio. Example
以下、 本発明を実施例により更に具体的に説明するが、 本発明は、 これらの実 施例に限定されるものではない。  Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.
(実施例 1) 培養に伴う配糖化反応による PN_ 5， 一 a_Gの生成 各種微生物の菌叢 1〜 2白金耳をかきとり、 0. 1%ピリ ドキシン塩酸塩（PN · HC 1 ) を含む下記表 1に示す培地 （5mLZTu b e) に入れ、 24〜3 0°C にて 3〜20日振盪あるいは静止培養した。培養後、培養液を 5分間煮沸した後、 遠心分離（15000rpm)を 1 0分間行った。得られた上清について下記表 2に示す条 件下で HP L C分析を行い、培養液中のピリ ドキシン一 5， - α—グルコシド（P N- 5 ' - a -G) 又はピリ ドキシン _ 4， 一 α—ダルコシド （ΡΝ— 4， - a 一 G) を定量し、 5 ' 選択率 （％) を下式によって求めた。 Below containing (Example 1) PN_ by glycosidation reactions associated with culture 5, scraped lawn 1-2 loopful of generating various microorganisms one a _G, 0. 1% pyridinium Dokishin hydrochloride (PN · HC 1) The cells were placed in the medium shown in Table 1 (5 mL Z Tube) and cultured at 24 to 30 ° C for 3 to 20 days with shaking or static culture. After the culture, the culture was boiled for 5 minutes and centrifuged (15000 rpm) for 10 minutes. The obtained supernatant was subjected to HP LC analysis under the conditions shown in Table 2 below, and pyridoxin-15, -α-glucoside (PN-5'-a-G) or pyridoxine_4 in the culture broth was analyzed. , One α-darcoside (ΡΝ-4, -a-1G) was quantified, and the 5 ′ selectivity (%) was determined by the following equation.
5， 選択率 （％) = (PN— 5， 一ひ一 Gのピーク面積 ZPN— 4， - a-G のピーク面積 +PN— 5， のピーク面積） X I 0 0 (培地組成） 5, selectivity (%) = (PN-5, peak area of G-1 ZPN-4,-peak area of aG + peak area of PN-5) XI 0 0 (Medium composition)
スクロース 2. 0%  Sucrose 2.0%
デキストリン 2. 0%  Dextrin 2.0%
ペプトン 1. 0%  Peptone 1.0%
酵母エキス 0. 05%  Yeast extract 0.05%
K2HP04 0 5% K 2 HP0 4 0 5%
KH2P04 0 1% KH 2 P0 4 0 1%
F e S 04 7H20 0 02% F e S 0 4 7H 2 0 0 02%
Mg S04 7H20 0 02% Mg S0 4 7H 2 0 0 02%
Mn S 04 nHaO 0 01 % Mn S 0 4 nH a O 0 01%
C a C 1 ¾ · 2H90 0 005( C a C 1 ¾2H 9 0 0 005 (
PN · HC 1 0. 1%  PNHC 1 0.1%
p H 7. 0 精製水 表 2  pH 7.0 Purified water Table 2
(HP LC条件）  (HP LC conditions)
カラム： Cosmosil 5C18AR (φ4.6X15匪）  Column: Cosmosil 5C18AR (φ4.6X15 marauder)
カラム温度：室温  Column temperature: room temperature
溶離液： 0.5%MeOH  Eluent: 0.5% MeOH
流速： 0.8mL/min  Flow rate: 0.8mL / min
検出： UV325nm 培養中に PNから 1. 5%以上の生成率で PN— 5 α— Gを生成し、 5 選択性が 75 %以上であった株を表 3に示した。 Detection: Table 3 shows the strains that produced PN-5α-G at a production rate of 1.5% or more from PN during UV325nm culture and had a 5-selectivity of 75% or more.
表 3 生成率 5' 選択率 m iiffl齒m Leiisonia aQuaOica. ττ rτπvj 110;7 ίi n U o. U Table 3 Generation rate 5 'Selectivity m iiffl teeth m Leiisonia aQuaOica. Ττ rτπvj 110; 7 ίin U o. U
Γ3·ΘΧ1 US 3· V61 ΤΤ7Π o Oj.  Γ3ΘΧ1 US 3 VV61 ΤΤ7Π o Oj.
协總齒 -SeUQOilO ΤΤ7Π 1 74. ¾ Π 协 Tooth -SeUQOilO ΤΤ7Π 1 74. ¾ Π
L/ryp iococcus a> UtJOO 11 O . Π W  L / ryp iococcus a> UtJOO 11 O .Π W
Cryptococcus terreus ?π Π797 . ¾  Cryptococcus terreus? Π Π797. ¾
OriO US 1DU 3· ΤΤ7Π AQAQ f ,
Figure imgf000013_0001
OriO US 1DU 3 ΤΤ7Π AQAQ f,
Figure imgf000013_0001
f 1' 11 A Q 1  f 1 '11 A Q 1
Coriolus puoescens ru Q y7 /S¾9 ^ .0  Coriolus puoescens ru Q y7 / S¾9 ^ .0
orio us Tinico οι* Tu Γnυ fiQfi^ 1 o 1 QV OJT O U VGJTfc* O 1 ζ  orio us Tinico οι * Tu Γnυ fiQfi ^ 1 o 1 QV OJT O U VGJTfc * O 1 ζ
jCiuro iuin a^Druni 77 4.  jCiuro iuin a ^ Druni 77 4.
rU «¾ 11 . 1  rU «¾ 11.1
u iioae ina &ρρ ansiiuni ΤΤ r7Π u 3 < 1147 A 1 o Q 1  u iioae ina & ρρ ansiiuni ΤΤ r7Π u 3 <1147 A 1 o Q 1
loc SQiuin au eum r yuo 7fi 7 β loc saiuiii virens ΛΜ OUOx 1 Q Ό ηΘ丄 icosty丄 um nigricans οποι y 0  loc SQiuin au eum r yuo 7fi 7β loc saiuiii virens ΛΜ OUOx 1 Q Ό ηΘ 丄 icosty 丄 um nigricans οποι y 0
Mortierella alpina 0 t. O  Mortierella alpina 0 t. O
Pithomyces atro o丄 lvaceus rJ ς 7  Pithomyces atro o 丄 lvaceus rJ ς 7
ΌΌ0 0. OU. Ό  ΌΌ0 0. OU. Ό
Sen sophyllum comniune 1 Π U.9  Sen sophyllum comniune 1 Π U.9
Sen zophyllum commune Τ7ί AQ9Q o  Sen zophyllum commune Τ7ί AQ9Q o
o. q o  o. q o
Schi zophyl lum commune DOU 0. ό 07 Q  Schi zophyl lum commune DOU 0.ό 07 Q
Schizophyllum commune IAM 9006 16.6 90.2  Schizophyllum commune IAM 9006 16.6 90.2
Schizophyllum commune IAM 13042 9.2 81.1  Schizophyllum commune IAM 13042 9.2 81.1
Trametes orientalis IF0 6483 2.7 77.7  Trametes orientalis IF0 6483 2.7 77.7
Verticillium albo-atrum IF0 9435 9.2 92.4  Verticillium albo-atrum IF0 9435 9.2 92.4
Verticillium dahliae IF0 9765 5.3 90.8  Verticillium dahliae IF0 9765 5.3 90.8
Verticillium dahliae IF0 31024 10.1 86.0  Verticillium dahliae IF0 31024 10.1 86.0
Verticillium dahliae JCM 9509 23.2 92.8  Verticillium dahliae JCM 9509 23.2 92.8
Verticillium dahliae JCM 9510 22.4 92.1  Verticillium dahliae JCM 9510 22.4 92.1
Verticillium tricorpus IF0 31025 9.1 77.0  Verticillium tricorpus IF0 31025 9.1 77.0
JCM: Japan Collections of Microorganisms, Institute of Physical and Chemical Research, Wako, Japan JCM: Japan Collections of Microorganisms, Institute of Physical and Chemical Research, Wako, Japan
IF0: Institute for Fermentation, Osaka, Japan  IF0: Institute for Fermentation, Osaka, Japan
IAM: Institute of Molecular and Cellular Bioscience, The University of Tokyo, Japan  IAM: Institute of Molecular and Cellular Bioscience, The University of Tokyo, Japan
NCIMB: National Colellection of Industrial and Marine Bacteria, Ltd.  NCIMB: National Colellection of Industrial and Marine Bacteria, Ltd.
Aberdeen, Scotland Aberdeen, Scotland
NCTC: National Collection of Type Cultures, Central Public Health Laboratory, London, England NCTC: National Collection of Type Cultures, Central Public Health Laboratory, London, England
CBS: Centraalbureau voor Schimmelcultures, Baarn, The Netherland  CBS: Centraalbureau voor Schimmelcultures, Baarn, The Netherland
ATCC: American Type Culture Collection, Rockville, USA ATCC: American Type Culture Collection, Rockville, USA
(実施例 2) 菌体を用いた PN— 5， _a_Gの生成 (Example 2) was used bacteria PN-5, product of _ a _G
(1) 実験方法  (1) Experimental method
4%可溶性デンプン、 1 %エスサンミート（味の素）、 0. 1%K2HP04、 0. 05%KC 1、 0. 05%Mg S04 · 7H20、 0. 01%F e SO4 - 7H2 0、 0. 1%PN · HC 1を含む培地 1 5 OmLを入れた 50 OmL容三角フラ スコで、 Verticillium albo-atrum IF09435を 25 °Cにて 5〜 7日間振とう培養 した。 4% soluble starch, 1% S. San Meat (Ajinomoto), 0. 1% K 2 HP0 4, 0. 05% KC 1, 0. 05% Mg S0 4 · 7H 2 0, 0. 01% F e SO4 - 7H Verticillium albo-atrum IF09435 was shake-cultured at 25 ° C for 5 to 7 days in a 50 OmL triangular flask containing 15 OmL of a medium containing 20 and 0.1% PN • HC1.
2% (0. 1M) PN . HC 1、 2%デキストリン、 0. 1Mリン酸カリウム 緩衝液 （pH6. 5 ； K-PB) 又は 0. 1Mホウ酸塩緩衝液 （四ホウ酸ニナト リウム水溶液にホウ酸を加え pH5. 0に調整して調製） 1. 2mLにフラスコ 培養菌体 lmL分菌体 （湿重量 50〜108mg) を懸濁し、 pH5. 0〜6. 5に維持しながら 40 °Cに加温した。  2% (0.1 M) PN.HC1, 2% dextrin, 0.1 M potassium phosphate buffer (pH 6.5; K-PB) or 0.1 M borate buffer (in aqueous sodium sodium borate solution) Add boric acid to adjust the pH to 5.0) 1. Suspend 1 mL of the bacterial cells in a flask (wet weight: 50 to 108 mg) in 2 mL and maintain the pH at 5.0 to 6.5 at 40 ° C. Was heated.
(2) 結果  (2) Result
反応 1 8時間後の反応液について実施例 1と同様にして HP LC分析を行い、 PN-5' — a— Gを定量した。 結果を表 4に示す。 表 4 生成率 （％) 5' 選択率 （％) Eighteen hours after the reaction, the reaction solution was subjected to HP LC analysis in the same manner as in Example 1 to quantify PN-5'-a-G. Table 4 shows the results. Table 4 Generation rate (%) 5 'Selectivity (%)
Vertici丄 lium albo-atrum IF09435 K-PB 36. 77.5 Vertici 丄 lium albo-atrum IF09435 K-PB 36.77.5
BB 35.4 97.7  BB 35.4 97.7
K-PB ：リン酸カリゥム緩衝液  K-PB: potassium phosphate buffer
BB ：ホウ酸塩緩衝液 表 4に示されるように、 反応液にリン酸カリゥム緩衝液を用いた場合に比べ、 ホウ酸塩緩衝液を用いた場合は 5 ' 選択率を 9 7 %以上にまで高めることができ た。 BB: borate buffer As shown in Table 4, the 5 'selectivity was increased to 97% or more when the borate buffer was used, as compared with the case where the potassium phosphate buffer was used for the reaction solution.
(実施例 3 ) 菌体を用いた P N—5 '— α— G生成反応におけるホゥ酸の効果 実施例 2の方法に準じて Verticillium dahliae JCM 9510、 Verticillium dahliae 丄 FO 9765、 schizophyllum commune IAM 9006及ぴ Coriolus pubescens IFO 9782の各種微生物による P N— 5，一 a— G生成におけるホウ酸添加の効果を調 ベた。 結果を表 5に示す。 いずれの菌株においても、 反応液にリン酸カリウム緩 衝液を用いた場合と比べ、 ホウ酸塩緩衝液を用いた場合の方が 5 ' 選択率が高か つた。 表 5 (Example 3) Effect of boric acid on PN-5'- α -G production reaction using bacterial cells Verticillium dahliae JCM 9510, Verticillium dahliae illFO 9765, schizophyllum commune IAM 9006 and the like according to the method of Example 2. The effect of boric acid addition on the production of PN-5,1-a-G by various microorganisms of Coriolus pubescens IFO 9782 was investigated. Table 5 shows the results. In all strains, the 5 'selectivity was higher when borate buffer was used than when potassium phosphate buffer was used as the reaction solution. Table 5
5 ' 選択率 （％) 5 'selectivity (%)
Verticillium dahliae JCM 9510 K-PB 89. 7  Verticillium dahliae JCM 9510 K-PB 89.7
BB 98. 3  BB 98. 3
Verticillium dahliae IFO 9765 K-PB 87. 3  Verticillium dahliae IFO 9765 K-PB 87.3
BB 98. 4  BB 98. 4
Schizophyllum commune IAM 9006 K-PB 90. 9  Schizophyllum commune IAM 9006 K-PB 90.9
BB 97. 5  BB 97.5
Coriolus pubescens IFO 9782 K-PB 77. 4  Coriolus pubescens IFO 9782 K-PB 77.4
BB 98. 3  BB 98. 3
K-PB ：リン酸カリゥム緩衝液  K-PB: potassium phosphate buffer
BB ：ホウ酸塩緩衝液 BB: borate buffer
(実施例 4 ) 酵素による P N— 5 ， — G生成反応におけるホウ酸の効果 以下の試験において、 酵素としてひ一ダルコシダーゼはシグマ社製 rice由来 G9259, トランスダルコシダーゼは天野製薬製 Aspergillus niger由来トランスグ ルコシダーゼ L「ァマノ」、 C G T a s eは天野製薬製 Bacillus macerans由来コ ンチザィムを用いた。 また、 ピリ ドキシン塩酸塩 （PN.HC1) は、 第一ファインケ ミカル （株） 製、 デキストリンは、 松谷化学工業 （株） TK- 16を用いた。 なお、 HPLCの分析条件は実施例 1に記載の条件にて行つた。 (Example 4) Effect of boric acid on enzymatic PN-5, -G production reaction In the following tests, Hi-Darcosidase was used as an enzyme in rice, G9259 from rice manufactured by Sigma, and trans-dalcosidase was used as trans-glucosidase from Aspergillus niger manufactured by Amano Pharmaceutical. L Amano and CGT ase are from Bacillus macerans Anchisym was used. Pyridoxine hydrochloride (PN.HC1) was used by Daiichi Fine Chemical Co., Ltd., and dextrin was used by Matsutani Chemical Industry Co., Ltd. TK-16. The HPLC analysis was performed under the conditions described in Example 1.
( 1 ) 酵素による P N配糖体生成反応と緩衝液の種類の比較 (1) Enzyme-forming reaction of PN glycosides and comparison of buffer types
下記表 6の組成を有する反応液をガラス製サンプル管に入れ、 アルミホイルで 遮光し、 30°Cの振盪恒温槽でインキュベートし、 反応させた。  The reaction solution having the composition shown in Table 6 below was placed in a glass sample tube, protected from light with aluminum foil, incubated in a shaking thermostat at 30 ° C, and reacted.
ここで、 酢酸緩衝液は酢酸ナトリゥム緩衝液に水酸ィ匕ナトリウムを加えて ρ H を 7.0に調整することによつて調製し、ホゥ酸塩緩衝液はホゥ酸にて p Hを 7. 0に調整することによつて調製した。 反応の結果を表 7に示す。 表 6 a—グルコシダーゼ （SIGMA) 20U/mL  Here, the acetate buffer was prepared by adding sodium hydroxide to sodium acetate buffer to adjust ρ H to 7.0, and the borate buffer was adjusted to 7.0 with boric acid. Prepared by adjusting Table 7 shows the results of the reaction. Table 6 a—Glucosidase (SIGMA) 20U / mL
PN · HC 1 0.18 M  PNHC1 0.18 M
TK一 1 6 (デキストリン） 8 %  TK-1 16 (dextrin) 8%
C a C 1 2 0.5 niM  C a C 1 2 0.5 niM
酢酸緩衝液又はホゥ酸塩緩衝液 (j)H7. 0 ) 0.18 M  Acetate buffer or borate buffer (j) H7.0 (0.18 M)
30°C、 p H6.5-7.0 表 7 反応時間（時間）  30 ° C, pH 6.5-7.0 Table 7 Reaction time (hour)
0 23 72  0 23 72
PN 100 93.05 88.03  PN 100 93.05 88.03
PN- 4'- α - G 0 1.95 3.47  PN- 4'- α-G 0 1.95 3.47
PN-5'-a-G 0 5 8.5  PN-5'-a-G 0 5 8.5
ホウ酸塩緩衝液 PN 100 98.2 96.8  Borate buffer PN 100 98.2 96.8
PN-4' -a-G 0 0 0.31  PN-4 '-a-G 0 0 0.31
PN-5'-a-G 0 1.72 3.06 表 7に示すように、 酢酸緩衝液に比べホゥ酸塩緩衝液を使用した場合、 生成物 の PN— 4， 一 a— Gと PN— 5， 一 α— Gの比率は、 29： 71力 ら 9： 91と、 Ρ Ν— 5， 一 α— Gの生成比率が明らかに向上した。 PN-5'-aG 0 1.72 3.06 As shown in Table 7, when the borate buffer was used as compared to the acetate buffer, the ratio of the PN-4,1-a-G to PN-5,1-α-G of the product was 29:71 9:91, the production ratio of Ρ Ν—5, one α —G was clearly improved.
(2) 酵素による Ρ Νの配糖化反応におけるホゥ酸塩緩衝液濃度の影響 (2) Effect of borate buffer concentration on glycosylation of Ν 配 by enzyme
CGT a s e (コンチザィム）とトランスダルコシダーゼを用い、 下記表 8の糸且 成を有する反応液において、 ホウ酸塩緩衝液濃度を基質 PNの等モル、 1 2、 1 /4とかえ、 反応を行った。 表 8  The reaction was carried out using CGTase (Contizym) and transdarcosidase in the reaction solution having the composition shown in Table 8 below, changing the concentration of the borate buffer to equimolar, 12, 1/4 of the substrate PN. Was. Table 8
CGT a s e (コンチザィム） （天野） 120 U/mL CGT a se (Conti-Zim) (Amano) 120 U / mL
又はトランスダルコシダーゼ（天野） 85000単位 ZmL  Or trans-dalcosidase (Amano) 85000 units ZmL
PN · HC 1 0.15  PNHC1 0.15
TK- 16 (デキストリン） 8 %  TK-16 (dextrin) 8%
C a C 12 0.5 mM C a C 1 2 0.5 mM
酢酸緩衝液又はホウ酸塩緩衝液 (p_H7.4)_ 0.15 M  Acetate buffer or borate buffer (p_H7.4) _ 0.15 M
30°C、 p H6.5-7.0 結果を表 9 (CGT a s e) 及び表 10 (トランスダルコシダーゼ） に示す。 なお、 PN—オリゴはピリ ドキシンに 2分子以上のグルコースが α結合した配糖 体を示す。 30 ° C, pH 6.5-7.0 The results are shown in Table 9 (CGTase) and Table 10 (transdarcosidase). PN-oligo is a glycoside in which two or more glucose molecules are α-linked to pyridoxine.
9 9
(CGT a s e) 反応時間 （時間）(CGT a se) Reaction time (hour)
0 25 64 1440 25 64 144
PN 100 81.72 73.89 60.76 酢酸 PN-4'-a-G 0 3.54 6.53 10.02PN 100 81.72 73.89 60.76 Acetic acid PN-4'-a-G 0 3.54 6.53 10.02
0.15M PN-5' -a-G 0 6.56 10.25 16.66 0.15M PN-5 '-a-G 0 6.56 10.25 16.66
PN -ォリ ゴ 0 7.69 9.33 12.55 PN-Oligo 0 7.69 9.33 12.55
PN 100 99.35 98.74 98.02 ホウ酸 PN-4' -a-G 0 0.12 0.21 0.34PN 100 99.35 98.74 98.02 Boric acid PN-4 '-a-G 0 0.12 0.21 0.34
0.15M PN-5' -a-G 0 0.53 1.05 1.63 0.15M PN-5 '-a-G 0 0.53 1.05 1.63
PN -ォリゴ 0 0 0 0 PN-Oligo 0 0 0 0
PN 100 97.31 94.45 92.3 ホウ酸 PN-4' -a-G 0 0.6 1.14 1.8PN 100 97.31 94.45 92.3 Boric acid PN-4 '-a-G 0 0.6 1.14 1.8
0.075M PN-5' -a-G 0 1.54 2.76 4.24 0.075M PN-5 '-a-G 0 1.54 2.76 4.24
PN -ォリ ゴ 0 0.55 1.65 3.75 PN-Oligo 0 0.55 1.65 3.75
PN 100 87.56 79.98 72.38 ホウ酸 PN-4' -a-G 0 2.34 4.2 6.99PN 100 87.56 79.98 72.38 Boric acid PN-4 '-a-G 0 2.34 4.2 6.99
0.0375M PN-5' -a-G 0 4.69 8.4 12.34 0.0375M PN-5 '-a-G 0 4.69 8.4 12.34
PN -ォリゴ 0 5.38 7.41 8.29  PN-Oligo 0 5.38 7.41 8.29
0 0
(トランスダルコシダーゼ) 緩衝液 反応時間（時間）  (Transdalcosidase) Buffer Reaction time (hours)
0 22 70  0 22 70
PN 100 94.13 93.19 酢酸 PN-4'-a-G 0 2.99 3.63  PN 100 94.13 93.19 Acetic acid PN-4'-a-G 0 2.99 3.63
0.15M PN-5' -a-G 0 2.88 3.18  0.15M PN-5 '-a-G 0 2.88 3.18
PN-ォリ ゴ 0 0 0  PN-origo 0 0 0
PN 100 99.46 98.88 ホウ酸 PN-4' -a-G 0 0 0  PN 100 99.46 98.88 Boric acid PN-4 '-a-G 0 0 0
0.15M PN-5' -a-G 0 0.54 1.11  0.15M PN-5 '-a-G 0 0.54 1.11
PN -ォリゴ 0 0 0  PN-Oligo 0 0 0
PN 100 99.01 97.7 ホウ酸 PN-4' -a-G 0 0.26 0.36  PN 100 99.01 97.7 Boric acid PN-4 '-a-G 0 0.26 0.36
0.075M PN-5' -a-G 0 0.74 1.82  0.075M PN-5 '-a-G 0 0.74 1.82
PN -ォリゴ 0 0 0 CGT a s eについて反応 144時間後における P N— 4， - α-G ： PN- 5， 一 α— Gは、 ΡΝと等量 (0.15M)の酢酸緩衝液を添加した場合は 38： 62であ るのに対し、 ΡΝの 1ノ 2等量（0.075Μ)のホウ酸塩緩衝液を添加した場合は 29： 71、 ΡΝの等量（0.15M)のホウ酸塩緩衝液を添加した場合は 17： 83であった。 ま た、 トランスダルコシダーゼについて反応 70時間後における ΡΝ_ 4， -α- G : PN— 5， 一 ひ一 Gは、 PNと等量 (0.15M)の酢酸緩衝液を添加した場合は 53： 47であるのに対し、 P Nの 1 / 2等量（0.075M)のホゥ酸塩緩衝液を添加した 場合は 17:83、 P Nの等量 (0.15M)のホゥ酸塩緩衝液を添加した場合は 0： 100 (ΡΝ-4，- α - Gの検出限界 0.1%であるので 8 ： 92以上） となった。 PN-Oligo 0 0 0 PN-4, -α-G after 144 hours of reaction for CGTase: PN-5, one α-G is 38:62 when an acetate buffer equivalent to ΡΝ (0.15M) is added. On the other hand, when 1 712 equivalents of 緩衝 (0.075Μ) borate buffer was added, the ratio was 29:71. When 等 equivalent (0.15M) of borate buffer was added, 17 17. : 83. In addition, ト ラ ン ス _4, -α-G: PN-5,1H-1G after 70 hours of reaction with transdarcosidase was 53:47 when an acetate buffer equivalent to PN (0.15M) was added. 17:83 when a 1/2 equivalent volume of PN (0.075M) borate buffer was added, and when a 1% equivalent of PN (0.15M) borate buffer was added Was 0: 100 (8:92 or more because the detection limit of ΡΝ-4, -α-G is 0.1%).
上記結果は、 ΡΝ分子とホウ酸分子がキレートすることによるものと考えられ る。 産業上の利用可能性  The above results are considered to be due to chelation of the ΡΝ molecule and the boric acid molecule. Industrial applicability
本発明によれば、 ピリ ドキシン又はその塩からピリ ドキシン一 5' — 一ダル コシドを容易にかつ効率的に製造することができる。  According to the present invention, pyridoxine-5′-dalcoside can be easily and efficiently produced from pyridoxine or a salt thereof.

Claims

請求 の 範 囲 The scope of the claims
1 . ピリ ドキシンの 5 ' 位を配糖ィ匕してピリ ドキシン一 5， 一 α _ダルコシドを 製造する方法であって、 下記のいずれかの属に属し、 ピリ ドキシンの 5， 位を選 択的に配糖化してピリ ドキシン一 5 ' — α—ダルコシドを生成する能力を有する 微生物の菌体、 その培養物、 又は菌体処理物でピリ ドキシン又はその塩を処理す る工程を含む方法。 1. A method for producing pyridoxine-1,5- α- darcoside by glycosylation of the 5'-position of pyridoxine, which belongs to any of the following genera and selects the 5-position of pyridoxine. A method comprising the step of treating pyridoxine or a salt thereof with a microbial cell, a culture thereof, or a processed product of a microorganism having an ability to form glycosylated pyridoxin-1 5'-α-darcoside.
レイフソニァ （Leifsonia)属 Genus Leifsonia
ノ ェ =■ノ チノレス (Paenibacillus)属 No = genus Paenobacillus
シユードノ力ノレァィ了 (Pseudonocardia) Pseudonocardia
クリプトコッカス (Cryptococcus) f Cryptococcus f
コリォルス （Coriolus)属 Genus Coriolus
ユーロチウム (Eurotium)属 Genus Eurotium
フラムリナ （Flammulina)属 Genus Flammulina
力ノデノレマ (Ganoderma)属 Genus Ganoderma
グリオクラディウム （Gliocladium)属 Gliocladium genus
ヘリコステイノレム (Helicostylum)属 Helicostylum
モルティエレラ （Mortierella)属 Genus Mortierella
ピソマイセス （Pithomyces)属 Genus Pithomyces
シゾフィノレム (Schizophyllum)属 Genus Schizophyllum
トラメテス （Trametes)属  Genus Trametes
ベノレティシリウム (Vert ici Ilium) 属 Genus Verret ici Ilium
2 . ホゥ酸及び Z又はホウ酸塩の存在下で処理を行う請求の範囲第 1項に記載の 方法。  2. The method according to claim 1, wherein the treatment is performed in the presence of boric acid and Z or a borate.
3 . ピリ ドキシン又はその塩を配糖ィ匕酵素で処理してピリ ドキシン一"一ダルコ シドを製造する方法において、 ホウ酸及び Z又はホウ酸塩の存在下で処理を行い 3. In a method for producing pyridoxine-1 "-dalcoside by treating pyridoxine or a salt thereof with a glycosylation enzyme, the treatment is carried out in the presence of boric acid and Z or borate.
5 ' 位の配糖化の選択性を高める方法。 How to increase the selectivity of 5 'glycosylation.
4 . ピリ ドキシン又はその塩を配糖ィ匕酵素で処理してピリ ドキシン一 α—ダルコ シドを製造する方法において 5， 位の配糖化の選択性を高めるための試薬であつ て、 ホウ酸及び Z又はホウ酸塩を含む試薬。 4. Pyridoxine or its salt is treated with glycosylation enzyme to give pyridoxine-α-darco. A reagent containing boric acid and Z or borate for enhancing the selectivity of glycosylation at the 5-position in the method for producing a side.
PCT/JP2003/000543 2002-01-22 2003-01-22 PROCESS FOR PRODUCING PYRIDOXINE-5’-α-GLUCOSIDE WO2003062442A1 (en)

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