JP6156947B2 - Method for producing glycoside - Google Patents

Description

  The present invention relates to a method for producing a glycosyltransferase derived from a microorganism belonging to the genus Ensifer and a glucoside of a compound having an OH group using a culture of the microorganism, and particularly to a method for producing a glycoside of gingerol. .

  A glycoside is a compound in which a hemiacetal hydroxy group of a sugar is bound to an aglycone which is a non-sugar component. Glycosides are widely distributed in the living world, and as glycosides, sugarcane oil glycosides, which are the main components of the pungent taste of plant pigments, anthocyanins, flavones, and mustards, saponins (steroids, steroidal alkaloids, Known are triterpene glycosides), glycolipids, nucleosides, antibiotics, and the like. When it becomes a glycoside, its physical properties change, such as improvement in water solubility and stability compared to the compound before the sugar is bound.

  Glycosylation research is being conducted with the aim of improving the water solubility and stability of compounds and finding new functions. For example, it has been reported that ascorbic acid was glycosylated with α-glucosidase or cyclodextrin glucanotransferase (Non-patent Documents 1 and 2). Ascorbic acid is easily decomposed by heat, light, oxygen, etc., but can be stably present by glycosylation. Moreover, although menthol is a property which is easy to volatilize and is hard to melt | dissolve in water, it is disclosed that aroma is maintained and water solubility is improved by glycosylation (Patent Document 1). As a method for glycosylation of menthol, for example, a method for producing a menthol glycoside by enzymatic reaction using saccharides such as sucrose and maltose and menthol using α-glucosidase (Patent Document 2), Xanthomonas genus, Stenotrophomonas genus, Arthrobacter genus There has been proposed a method of producing with a microorganism having menthol glycosylation ability belonging to (Patent Document 3).

  Gingerol known as an essential oil component contained in the rhizomes of Zingiberaceae plants is known to have effects such as fat accumulation suppression (Non-patent Document 3), adiponectin production enhancement (Patent Document 4), It is used as a raw material for pharmaceuticals such as digestives, laxatives, antitussives and antacids, as a food for seasonings, soft drinks, alcoholic beverages, confectionery and the like, and as a fragrance for cosmetics.

  However, gingerol is hardly soluble in water and has a strong pungent taste and irritation. In addition, under heating or acidic conditions, gingerol is converted to gingerol or gingerone, and is less soluble in water, resulting in strong hotness.

  In order to solve such problems, there has been proposed a method (Patent Document 5) for obtaining a gingerol-containing water-soluble composition having high stability and high water solubility by combining gingerol or a gingerol-containing material and a heat-treated saccharide. . However, since the composition obtained by the method is caramel-like, the composition is limited to a specific application, and the ethanol remains, which causes problems such as difficulty in use for food and drink. In order to improve these problems, it was reported that gingerol was glycosylated using a glycosyltransferase derived from bacteria of the genus Halomonas, which improved the water solubility and stability of the obtained gingerol glycoside. (Patent Document 6). However, a method for producing gingerol glycosides from glycosyltransferases other than glycosyltransferases derived from the genus Halomonas is not known.

  Gingerol glycosides are easy to handle in foods and beverages, quasi-drugs, pharmaceutical industry, etc., and are highly versatile. In industrial use, gingerol derivatives including glycosides and efficient production methods thereof are required.

JP-A-61-83114 JP-A-9-224693 Japanese Patent Application Laid-Open No. 11-155591 JP 2011-1386 A JP 2008-56572 A JP 2013-112623 A

Biochim. Biophys. Acta., 1990, 1035, 44-50. Agric. Biol. Chem. 1991, 55, 1751-1756. J. et al. Med. Chem. , 2011, 54, 6295-6304

  An object of the present invention is to provide a glycosyltransferase for efficient glycosylation, a glycoside of a compound having an OH group, and a method for producing the glycoside of the compound. It is to provide a method for producing glycosyltransferase, gingerol glycoside, and gingerol glycoside which are saccharified.

  As a result of diligent research, the present inventors have reacted a compound having an OH group and a sugar donor such as maltose in the presence of a culture of a microorganism belonging to the genus Ensifer. Thus, it was found that a glycoside of a compound having an OH group can be synthesized. Moreover, it discovered that a gingerol glycoside was produced | generated by making the culture of the microorganisms which belong to Encipher genus contact with gingerol in presence of a sugar donor. The present invention has been accomplished based on these findings.

That is, the present invention is as follows.
(1) A glycosyltransferase derived from a microorganism belonging to the genus Ensifer, which has an activity of producing a glycoside of the compound in the presence of a compound having an OH group and a sugar donor.
(2) The glycosyltransferase according to (1) above, wherein the microorganism belonging to the genus Encipher is E. adhaerens.
(3) The glycosyltransferase according to (1) or (2) above, wherein the compound having an OH group is gingerol.
(4) A culture of a microorganism belonging to the genus Encipher for use in producing a glycoside of the compound in the presence of a compound having an OH group and a sugar donor.
(5) The microorganism culture of (4) above, wherein the microorganism belonging to the genus Encipher is Encipher adherence.
(6) The microorganism culture of (4) or (5) above, wherein the compound having an OH group is gingerol.
(7) The glycosyltransferase according to any one of (1) to (3) above or (4) to (4) above in the presence of a sugar donor in an extract containing a compound having an OH group or a compound having an OH group. (6) A process for producing a glycoside of a compound having an OH group, which comprises the step of allowing a culture of a microorganism to act.
(8) The method for producing a glycoside according to (7) above, wherein the compound having an OH group and the extract containing the compound having an OH group are gingerol and a plant extract containing gingerol, respectively.
(9) The glycoside of the above (7) or (8), wherein the glycoside of the compound having an OH group is a glycoside obtained by dehydrating condensation of glucose, maltose or maltooligosaccharide to the OH group of the compound. A method for producing a saccharide.
(10) The method for producing a glycoside according to any one of the above (7) to (9), wherein the glycoside is a gingerol glycoside represented by the following general formula (1).
(In the formula, R represents a glucosyl group, a maltosyl group or a glucosyl group of maltooligosaccharide, and n represents an integer of 2, 4, 6 or 8.)
(11) The gingerol glycoside represented by the general formula (1) is a gingerol glycoside in which glucose is bonded to the OH group of the side chain of 6-gingerol represented by the following formula (2) at the α-1 position. The method for producing a glycoside according to (10) above, wherein
(12) The gingerol glycoside represented by the general formula (1) is a gingerol glycoside represented by the following formula (3) in which maltose is bonded to the OH group of the side chain of 6-gingerol at the α-1 position. The method for producing a glycoside according to (10) above, wherein
(13) The method for producing a glycoside according to any one of (8) to (12), wherein the plant is ginger.
(14) Use of a culture of a microorganism belonging to the genus Encipher to produce a glycoside of the compound in the presence of a compound having an OH group and a sugar donor.

  The glycosyltransferase and the microorganism culture of the present invention can glycosylate a compound having an OH group, and the resulting glycosides have improved physical properties such as water solubility and stability than before glycosylation. To do. In addition, by glycosylation, a new bioactive function that was not found in the compound before glycosylation may appear or the activity may be enhanced. In particular, the glycosyltransferase and the culture of the microorganism are suitable for producing a gingerol glycoside. Since gingerol glycosides are easily dissolved in water and are more stable than gingerol, they can be used in foods, beverages, pharmaceuticals, cosmetics and the like. Furthermore, by using a gingerol glycoside, the conversion of gingerol to gingerol or gingerone can be suppressed, and the pungency derived from gingerol or gingerone can be reduced.

It is a figure which shows the analysis result of TLC which shows the production | generation of gingerol glycoside. 1 is a lane in which only 6-gingerol (Wako Pure Chemical Industries) is spotted, 2 is a lane in which 1 μl of reaction supernatant is spotted, 3 is a lane in which 2 μl of reaction supernatant is spotted, 4 is a lane in which 3 μl of reaction supernatant is spotted, 5 is a lane spotted with 4 μl of the reaction supernatant. It is a figure which shows the analysis result of HPLC which shows the production | generation of gingerol glycoside. It is a figure which shows the analysis result of LC-MS which shows the production | generation of a gingerol glycoside.

(Microorganisms belonging to Encifer genus)
In the present invention, the microorganism belonging to the genus Encipher refers to a microorganism that has been clarified to be included in the genus Encipher from the results of 16S rRNA gene base sequence analysis, and has conventionally been classified as, for example, the genus Sinorhizobium. Including things.

  Examples of microorganisms belonging to the genus Encipher include, for example, E. adhaerens (= S. morelense), Encipher Arboris, E. fredii, E. fredii, E.garamanticus, E.kostiensis, E.medicae, E.meliloti, E.saheli, Ensipher Teranga (E. terangae), Encifer xinjiangensis (E.xinjiangensis), Sinoamericanum (S.americanum), etc. can be mentioned.

  Specific strains belonging to the genus Encipher are described in, for example, the biological resources catalog of the National Institute of Technology and Evaluation (NITE), for example, Encipher Adherence NBRC100387, NBRC100388, NBRC108628, etc. Encipher Arboris NBRC100383, etc., Encipher Freddy NBRC100018, NBRC14780, etc., Encipher Galamanticus NBRC107849, etc. Telanga NBRC10035 etc., Encipher Xing Jian en-cis NBRC100018, etc., can be cited Sinorhizobium America Nam NBRC107163, etc., and these may be used may be used one share alone, also more than two strains together. Of these microorganisms belonging to the genus Encipher, microorganisms belonging to Encipher Adherence are preferred, and those belonging to Encipher Adherence can be easily obtained by anyone from among strains stored in NITE. .

  In addition, microorganisms belonging to the genus Encipher can be mutated by artificial mutation means using ultraviolet rays, X-rays, drugs, etc., and such mutant strains (mutant microorganisms) are also glycosyltransferases that are the subject of the present invention. As long as it has the activity described above, it can be used as a microorganism derived from the glycosyltransferase of the present invention.

  In addition, the microorganism belonging to the genus Encipher is transformed into a host cell (eg, Escherichia coli) that is transformed with a gene encoding the glycosyltransferase of the present invention derived from the microorganism belonging to the genus Encipher and expresses the glycosyltransferase of the present invention. A replacement) is also included for convenience. These recombinants can be prepared, for example, by the following method.

  The partial amino acid sequence of the glycosyltransferase of the present invention is determined by a conventional method, and an appropriate DNA probe is prepared based on the sequence. A glycosyltransferase gene can be cloned by screening the gene library of the encipher using the DNA probe. By comparing the base sequence of the obtained DNA with the information on the partial amino acid sequence, it can be confirmed whether or not the gene derived from the encipher has been obtained. The Encipher gene library can be prepared, for example, using a commercially available gene library preparation kit.

  Isolation of microbial DNA used for cloning, preparation of a genomic library, screening, and analysis of the base sequence can be performed by methods well known to those skilled in the art. For example, a purified glycosyltransferase is hydrolyzed, an oligonucleotide primer is synthesized based on the partial amino acid sequence information of the hydrolyzed fragment, and a partial sequence of the gene encoding the glycosyltransferase is obtained using a PCR reaction. Isolate. Subsequently, a DNA probe is prepared using this partial sequence, and an Encipher genomic library is screened, and a clone expressing the glycosyltransferase can be selected. From this selected clone, the sequence of the gene encoding the target protein can be clarified.

  As the host cell, for example, Escherichia coli, Bacillus subtilis, Bacillus brevis, etc. can be used, and the vector can be replicated in Escherichia coli. Plasmids such as pUC18, pUC19, pBR322, pGEM3, pGEM4, pUC110, pE194, pC194, etc. that can replicate in Bacillus subtilis, pNCMO2, pNY326, etc. that can replicate in Bacillus brevis can be used.

  The microorganism belonging to the genus Encipher is preferably cultured in a medium having a pH of about 5.0 to 8.0, a culture temperature of 20 to 40 ° C., and a culture time of 1 to 7 days. As a culture medium for microorganisms, any medium can be used as long as it is a medium used for normal microorganism culture. For example, a natural medium or a synthetic medium containing a carbon source, a nitrogen source, inorganic salts, and other nutrient substances can be used. Can be used. Examples of the shape of the medium include a liquid medium, a semi-fluid medium, a solid medium, and the like.

  Examples of the carbon source include glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, galactose, starch, molasses, sorbitol, glycerin and other sugars and sugar alcohols; acetic acid, citric acid, lactic acid, fumaric acid, maleic acid Or organic acids, such as gluconic acid; Alcohol, such as ethanol or propanol, etc. can be mentioned. A carbon source may be used individually by 1 type, and may mix and use 2 or more types. The density | concentration in the culture medium of these carbon sources is about 0.1-10 wt% normally.

Examples of the nitrogen source include nitrogen compounds such as ammonium chloride, ammonium sulfate ((NH ₄ ) ₂ SO ₄ ), inorganic or organic ammonium compounds such as ammonium nitrate and ammonium acetate, urea, aqueous ammonia, sodium nitrate and potassium nitrate. . Further, corn steep liquor, meat extract, peptone, NZ-amine, protein hydrolyzate, nitrogen-containing organic compounds such as amino acids, and the like can also be used. A nitrogen source may be used individually by 1 type, and may mix and use 2 or more types. The medium concentration of the nitrogen source varies depending on the nitrogen compound used, but is usually about 0.1 to 10 wt%.

Examples of the inorganic salts include monopotassium phosphate (KH ₂ PO ₄ ), dipotassium phosphate (K ₂ HPO ₄ ), magnesium sulfate (MgSO ₄ ), iron sulfate (FeSO ₄ ), sodium chloride, nitric acid first Examples thereof include iron, manganese sulfate, zinc sulfate, cobalt sulfate, and calcium carbonate. These inorganic salts may be used alone or in a combination of two or more. The culture medium concentration of inorganic salts varies depending on the inorganic salt used, but is usually about 0.01 to 1.0 wt%.

  Examples of the nutritional substance include meat extract, peptone, polypeptone, yeast extract, dry yeast, corn steep liquor, skim milk powder, defatted soy hydrochloride hydrolyzate, extracts of animals and plants or microbial cells, and degradation products thereof. it can. The medium concentration of the nutrient substance varies depending on the nutrient substance to be used, but is usually about 0.1 to 10 wt%.

  The pH of the medium is adjusted with an inorganic acid or alkali solution, an organic acid or alkali solution, ammonia, a pH buffer solution, or the like.

(Culture of microorganisms belonging to Encifer genus)
The culture of microorganisms belonging to the genus Encipher according to the present invention is a culture (fungi) having a limited use of producing a glycoside of the compound in the presence of a compound having an OH group and a sugar donor. Including itself). The culture also includes a treated product of the microorganism culture. The treated product includes wet cells separated from the culture supernatant, dried cells by freeze-drying or acetone treatment, and these cells. Examples include immobilized microorganisms, culture supernatants and the like bound to a carrier. In addition, in the culture of the microorganism belonging to the genus Encipher, a host cell such as Escherichia coli is transformed with the gene encoding the glycosyltransferase of the present invention derived from the microorganism belonging to the genus Encipher, Also included are cultures of such host cells that express.

(Glycosyltransferase)
The glycosyltransferase of the present invention is not particularly limited as long as it is an enzyme derived from a microorganism belonging to the genus Encipher having an activity of producing a glycoside of the compound in the presence of a compound having an OH group and a sugar donor. First, the glycosyltransferase-containing composition that has been separated and purified from the culture of the microorganism, the purified glycosyltransferase, and the immobilized sugar obtained by binding the glycosyltransferase-containing composition or the purified glycosyltransferase to a carrier Examples thereof include transferase.

  Separation and purification of glycosyltransferase from the culture of the microorganism can be performed by, for example, pulverizing microbial cells or culture supernatant using ultrasonic waves or glass beads, for example, ammonium sulfate precipitation, ion exchange (for example, DEAE cellulose column chromatography, Sepha Dex G-100 column chromatography, etc.), chelate affinity chromatography, gel filtration column chromatography, etc.

  As the carrier for immobilizing the dried microbial cells and the glycosyltransferase, any carrier can be used as long as it is used for immobilizing microorganisms and enzymes, for example, celite, diatomaceous earth, kaolinite. , Silica gel, molecular sieves, porous glass, activated carbon, calcium carbonate, ceramics and other inorganic carriers, ceramic powder, polyvinyl alcohol, polypropylene, acrylamide, carrageenan, chitosan, ion exchange resin, hydrophobic adsorption resin, chelate resin, synthetic adsorption resin, etc. The organic polymer of these can be mentioned. Examples of the method for immobilizing microorganisms or enzymes on the carrier include known methods such as an adsorption method, an ion binding method, a covalent bonding method, and a biochemical specific binding method. When an immobilized glycosyltransferase is used, it can be used repeatedly for batch production or continuously for production of glycosides.

  Furthermore, the glycosyltransferase of the present invention also includes a glycosyltransferase expressed in a recombinant obtained by transforming E. coli or the like with the gene encoding the glycosyltransferase of the present invention. In addition, since peptides or proteins are generally known to have the same activity even if one or several amino acids in the amino acid sequence are deleted, substituted or added, these deletions The activity of the glycosyltransferase that is the subject of the present invention, even if the protein is expressed from a base sequence that encodes a substituted or added amino acid sequence or a gene that is encoded by a base sequence that differs due to codon degeneracy. Can be used as long as it encodes a protein having the above, and the expressed protein is included in the glycosyltransferase of the present invention.

  The glycosyltransferase gene can be expressed in the recombinant obtained by transforming a host cell with an appropriate vector into which an appropriate promoter or a sequence involved in expression is introduced. Therefore, the glycosyltransferase of the present invention can be produced in large quantities by such a gene recombination technique.

(Compound having OH group)
The compound having an OH group in the present invention is not particularly limited as long as it is a compound having one or more OH groups that can be glycosylated by the glycosyltransferase or microorganism culture of the present invention. And a compound having a phenolic OH group. Examples of the compound having an alcoholic OH group include primary alcohols, secondary alcohols, and tertiary alcohols, and primary alcohols and secondary alcohols are preferable.

  Examples of the compound having an alcoholic OH group include aliphatic alcohols such as 1-butanol, 2-butanol, 1-nonanol, and 5-nonanol; monoterpene compounds having a hydroxy group such as geraniol and linalool; digitoxigenin, salsasaponigen, Steroids such as digitogenin; alkaloids such as solanidine; gingerol and the like.

  Examples of compounds having a phenolic OH group include phenols such as hydroquinone, salicyl alcohol, phloretin, pyrocatechol, and phenol; coumarins such as esculetin; flavonoids such as apigenin, daidzein, quercetin, hesperetin, and naringenin; Chalcones; anthocyanidins such as pelargonidin, cyanidin, and delphinidin; anthraquinones such as alizarin; indoles such as indoxyl; and alkaloids such as dopamine and adrenaline.

  As the compound having an alcoholic OH group or the compound having a phenolic OH group, a commercially available product can be used in addition to an extract obtained from a plant-derived raw material by a known method. Moreover, in this invention, the compound which has OH group may be provided with the form of the compound which has OH group, and may be provided with the form of the extract containing the compound which has OH group. For example, when it is a plant-derived raw material, the extract containing a compound having an OH group crushes, squeezes, powders, or pastes the entire plant or leaves, stems, flowers, pulp, roots, etc. After processing, etc., it can be obtained by performing an extraction process such as hot water extraction, ethanol extraction, supercritical extraction or the like as necessary.

  Among these compounds having an alcoholic OH group and compounds having a phenolic OH group, compounds having an alcoholic OH group are preferable, and gingerol is more preferable. Further, the compound having an OH group may be a natural product or a synthetic product.

  Examples of the gingerol include 6-gingerol, 4-gingerol, 8-gingerol, 10-gingerol, 3S, 5S-6-gingerdiol, 3R, 5S-6-gingerdiol, and the like. Good. As the gingerol, those extracted from a plant belonging to the family Gingae (Zingiberaceae) containing gingerol, preferably a plant such as ginger (Zingiber officinale) can be suitably used.

(Sugar donor)
The sugar donor is not particularly limited as long as the glycoside can be produced in the presence of the enzyme. For example, glucose; maltose; maltotriose, maltotetraose, maltopentaose, maltohexaose. Malto-oligosaccharides such as maltoheptaose and maltooctaose; dextrin; cyclodextrin; starch hydrolyzate called α-glucan such as amylose; or starch and mixtures thereof can be used as a sugar donor. The sugar donor may be a commercially available product, or a product obtained by allowing a hydrolase such as α-amylase, β-amylase, or isoamylase to act on starch or a starch degradation product.

(Glycosides)
The glycoside in the present invention is not particularly limited as long as the hemiacetal OH group of the sugar is bonded to a non-sugar component. For example, the phenols, coumarins, flavonoids, chalcones, anthocyanidins, Examples include glycosides such as anthraquinones, indoles, steroids, alkaloids, aliphatic alcohols, monoterpene compounds, and gingerol.

  For example, gingerol glycoside is represented by the following general formula (1).

(In the formula, R represents a glucosyl group, a maltosyl group or a glucosyl group of maltooligosaccharide, and n represents an integer of 2, 4, 6 or 8.)

  Among the gingerol glycosides represented by the general formula (1), a gingerol glycoside in which glucose is bonded to the OH group of the side chain of 6-gingerol represented by the following formula (2) at the α-1 position. Or gingerol glycoside in which maltose represented by the following formula (3) is bonded at the α-1 position.

(Manufacture of glycosides)
Examples of the method for producing a glycoside of the present invention include a method in which a glycosyltransferase of the present invention or a culture of a microorganism is added to a mixture of a compound having an OH group and a sugar donor to cause a glycosyltransferase reaction. it can. When the compound having an OH group is water-insoluble, it is usually separated from the aqueous phase. Therefore, it is preferable to carry out the reaction while stirring with an agitator, a stirrer or the like, or using a rotator or the like. An emulsifier may be mixed in the reaction solution. In addition, when the compound having an OH group is gingerol, gingerol glycoside is obtained by contacting gingerol or a plant extract containing gingerol with a sugar donor in the presence of the glycosyltransferase of the present invention or a microorganism culture. Can be manufactured.

  In the production of glycosides using the glycosyltransferase of the present invention, the amount of glycosyltransferase used and the reaction conditions greatly affect the production efficiency, so the reaction conditions such as the appropriate enzyme amount and reaction time should be selected. is important. Usually, from the viewpoint of economy, it is preferable to select an added amount of the enzyme so that the reaction can be completed in about 1 to 100 hours. In addition, examples of conditions under which the glycosyltransferase of the present invention sufficiently acts include conditions selected from the range of pH 5 to 10, temperature 15 to 50 ° C.

  The concentration of the glycosyltransferase of the present invention is usually 0.1 to 100 U per 1 g of sugar donor, and preferably 0.2 to 50 U per 1 g of sugar donor. 1 U represents the amount of enzyme that promotes the chemical reaction of 1 μmol of substrate per minute.

  In the production method of the present invention, the concentration of the compound having an OH group in the enzyme reaction solution is usually 1 w / v% or more, preferably 2 to 30 w / v%, and the sugar donor concentration (w / v ) Is preferably in a concentration range of 0.5 to 30 times that of the compound having an OH group.

  The reaction between the compound having an OH group and the sugar donor may be performed in the presence of a solvent. The solvent used in the reaction may be any kind and concentration range that does not affect the reaction. Specific examples include methanol, DMSO, 2-propanol, ethanol, and the like that can be usually used. These solvents may be used alone or in combination of two or more.

  In addition, the glycoside in the production method of the present invention can also be produced by contacting the culture of the microorganism with a compound having an OH group in the presence of a sugar donor. “Contacting a microorganism culture” means adding or incubating a compound having an OH group and a sugar donor to a medium in which the microorganism is cultured, or, for example, immobilizing a microorganism having an OH group and sugar donation. Refers to the body solution flowing down and contacting. In that case, the microorganism may or may not be present in the medium, and the “microorganism culture” includes, as described above, dried cells and crushed material of the microorganism, the sugar transfer Host cells transformed with the gene of the enzyme, dried cells and disrupted products of the host cells, purified glycosyltransferases, immobilized products thereof, and the like are included. In addition, when the medium contains saccharides such as glucose, fructose, sucrose, mannose, maltose, mannitol, xylose, galactose, starch, molasses, sorbitol, glycerin, the saccharide can be used as a sugar donor.

  In the state where the glycoside and the sugar donor produced as described above coexist, by further causing another glycosyltransferase to act, the glucose polymerization degree of the glycoside is set to 2 or more, or other than glucose Of course, it is possible to transfer the sugar of the glucosyl group to the glucosyl group of the glycoside, and the resulting substance is expected to be more water-soluble than monoglucoside.

  The reaction can be stopped by inactivating the contained enzyme by heating the mixture or lowering the pH.

  In addition to glycosides, the mixture containing glycosides produced as described above contains unreacted compounds having OH groups, sugar donors, and in some cases, enzymes and reaction by-products such as glucose. Since it is mixed, α-amylase (EC number: EC.3.2.1.1), β-amylase (EC number: EC.3.2.1.2), glucoamylase (EC number: EC.3.2.1.1) EC number: EC 3.1.1.3) and the like can be subjected to hydrolysis treatment to reduce the molecular weight of the remaining sugar donor.

  Since OH group compounds and glycosides have different solubility in water, they can be separated into an oil phase and an aqueous phase by an extraction operation, and glycosides are dissolved in a more hydrophilic solvent. Further, a purified product of high-purity glycoside can be easily obtained by further separating and purifying this hydrophilic solvent by a method such as ion exchange column chromatography, gel filtration chromatography, or hydrophobic chromatography.

  The solvent that can be used for the extraction operation is not particularly limited, but the glycoside in the present invention, for example, gingerol glycoside, is insoluble in a hydrocarbon solvent such as hexane, water, an ether solvent such as THF, and ethyl acetate. Since it is soluble in an ester solvent and gingerol is insoluble in water, it is preferable to use these solvents in combination.

  Specifically, for example, after removing microorganisms from the reaction solution as necessary, unreacted gingerol and glycosides are extracted with an ester solvent or an ether solvent, and the extract is dried, It is preferable to extract and remove the gingerol of the reaction with a hydrocarbon solvent and dissolve the residue in an ester solvent as necessary to obtain a glycoside. Further, after removing microorganisms from the reaction solution as necessary, gingerol may be extracted and removed first with a hydrocarbon solvent. The details of the operation procedure may be determined from the amount of solvent used, the oily substance contained in the raw material, the glycoside purity in the ester solvent, and the like.

  Examples of the ester solvent that can be used include ethyl acetate and the like, and examples of the ether solvent include diethyl ether and THF, among which ethyl acetate is preferable. Examples of the hydrocarbon solvent include hexane and chloroform. Among these, hexane is preferable. In addition, after extracting unreacted gingerol and glycosides with an ester solvent from the reaction solution from which the microorganisms have been removed, a hydrocarbon solvent such as hexane, which is a poor solvent for gingerol glycosides, is added to obtain gingerol glycosides. Priority crystallization is also possible.

The method for purifying glycosides can be applied not only to gingerol glycosides produced using microorganisms in the present invention, but also to gingerol glycosides produced using cultures of other microorganisms.
The other microorganism is not particularly limited as long as it is a microorganism having an activity to produce a glycoside of gingerol in the presence of gingerol or a plant extract containing gingerol and a sugar donor. Specifically, for example, a microorganism (for example, Escherichia coli) obtained by transforming a glucosidase gene derived from a microorganism belonging to Halomonas described in Patent Document 6.

  Thus, according to the present invention, in the presence of gingerol or a plant extract containing gingerol and a sugar donor, a culture of a microorganism having an activity to produce a glycoside of gingerol or a treated product thereof is brought into contact with gingerol glycoside. A method for producing gingerol glycosides is provided, which includes a step of producing a body and a step of extracting the produced gingerol glycoside with an ester solvent.

  A mixture of the above glycoside, a hydrolyzed product of the mixture, an enzyme-inactivated product of the mixture, a purified product of the mixture, a dried product, a powdered product, etc. It can be used for foods and drinks, cosmetics, quasi drugs or pharmaceuticals as a composition for pharmaceuticals, quasi drugs or pharmaceuticals.

  Hereinafter, the present invention will be described in detail with reference to examples. However, the present invention is not limited to the following examples.

[Example 1] Production of gingerol glycoside

(1) Preparation of agar medium Maltose (manufactured by Nippon Shokuhin Kako) 5 g, yeast extract (manufactured by Becton Dickinson) 100 mg, (NH ₄ ) ₂ SO ₄ (manufactured by Wako Pure Chemical Industries) 2 g, KH ₂ PO ₄ (manufactured by Kanto Chemical) ) 1 g, 1 g of K ₂ HPO ₄ (manufactured by Wako Pure Chemical Industries), 0.2 g of MgSO ₄ .7H ₂ O (manufactured by Kanto Chemical), 0.01 g of FeSO ₄ · 7H ₂ O (manufactured by Kanto Chemical) and agar BA for medium 15 g of -10 (manufactured by Ina Food Industry) was weighed out, transferred to a 2 L beaker, dissolved in 1 L of distilled water, dissolved, and adjusted to pH 7.0 with NaOH. The solution was sterilized in an autoclave at 121 ° C. for 20 minutes, and then dispensed in 15 ml portions into a petri dish in a clean bench. The petri dish lid was slightly opened and allowed to stand for 20 minutes. After cooling, it was used as an agar medium.

(2) Production of liquid medium 50 g of maltose (manufactured by Nippon Shokuhin Kako), 2 g of polypeptone (manufactured by Wako Pure Chemical Industries), 2 g of yeast extract (manufactured by Becton Dickinson), (NH ₄ ) ₂ SO ₄ (manufactured by Wako Pure Chemical Industries) Weigh ₂ g, 1 g of KH ₂ PO ₄ (manufactured by Kanto Chemical), 1 g of K ₂ HPO ₄ (manufactured by Wako Pure Chemical Industries), 0.2 g of MgSO ₄ .7H ₂ O (manufactured by Kanto Chemical), transfer to a 2 L beaker and distill. 1 L of water was added, dissolved, and adjusted to pH 7.0 with NaOH. 50 ml of the solution was charged into a 200 ml baffled Erlenmeyer flask, sterilized in an autoclave at 121 ° C. for 20 minutes, cooled, and then used as a liquid medium.

(3) Cultivation and production of gingerol glycoside Encipher Adherence NBRC100387, NBRC100388, and NBRC108628 strains cultured at 25 ° C. for 2 to 3 days on the agar medium, respectively, were planted in 2 platinum ears in the liquid medium. And cultured with shaking at 25 ° C. and 160 rpm for 42 hours.
After the culture, 0.1 ml of a methanol solution containing 14 mg of 6-gingerol was added to the culture solution, and the reaction was started at 25 ° C. and 160 rpm. 24 hours after the addition of 6-gingerol, 5 ml of methanol was added, and then the cells were separated by centrifugation to obtain a reaction supernatant containing the produced gingerol glycoside.

[Example 2] Analysis of gingerol glycoside

  The reaction supernatant containing gingerol glycoside obtained in Example 1 was analyzed by TLC, HPLC and LC-MS.

(1) Analysis by TLC Using a TLC aluminum plate (Merk kieselgel 60 F254) as a TLC plate and 2-propanol: n-butanol: water = 2: 2: 1 as a developing solvent, 6-gingerol ( (Wako Pure Chemical Industries) only (lane 1), reaction supernatant 1 μL (lane 2), reaction supernatant 2 μl (lane 3), reaction supernatant 3 μl (lane 4), reaction supernatant 4 μl (lane 5) ,analyzed. After the development, 10% (v / v) methanol sulfate was sprayed and colored by heating to confirm the reaction product. As preparations, 1 μL of a 1 w / v% aqueous solution of glucose (manufactured by Kanto Chemical) and maltose (manufactured by Nippon Shokuhin Kako) was used. The results of TLC analysis with the NBRC100387 strain are shown in FIG. Similar analysis results of TLC were obtained for the other two strains.

  One day after the reaction, it was confirmed that 6-gingerol was halved and a gingerol glycoside was produced.

(2) Analysis by HPLC COSMOSIL 5C18-MS-II 4.6 mm × 150 mm as an analytical column, methanol: water = 7: 3 as an eluent, and UV absorption at 280 nm under conditions of room temperature and flow rate 0.6 ml / min. Analysis was carried out. The results for the NBRC100387 strain are shown in FIG. Similar results were obtained for the other two strains.

  Also in HPLC, a peak was observed at an elution time of 8 to 9 minutes, and the production of gingerol glycoside was confirmed.

(3) Analysis by LC-MS An equal amount of ethyl acetate was added to the reaction supernatant and mixed well. After standing, the aqueous layer and the ethyl acetate layer were measured by LC-MS. Measurements were made at the Advanced Lipid Analysis Laboratory, Health Innovation Center, Graduate School of Health Sciences, Hokkaido University. The results with the NBRC100387 strain are shown in FIG. Similar results were obtained for the other two strains.

According to LC-MS analysis, an ammonium adduct ion (molecular formula: C ₂₃ H ₄₀ O ₉ N, theoretical value: 474.2698 [(M + NH ₄ ) ⁺ ]) obtained by measurement of the gingerol glycoside in which glucose is bonded to 6-gingerol: 474.2698) and an ammonium adduct ion of a gingerol glycoside in which maltose is bound to 6-gingerol (molecular formula: C ₂₉ H ₅₀ O ₁₄ N, theoretical value: 636.3226 [(M + NH ₄ ) ⁺ ], actually measured value: 636 3231) was observed. The observation of the above ion peak indicates that 6-gingerol and maltose formed a glycosidic bond to become a gingerol glycoside.

[Example 3] Production of alcoholic hydroxyl group glycoside

  The reaction was performed in the same manner as in Example 1 using 1-butanol, 5-nonanol, and 1-nonanol as the sugar receptor. Whether or not a glycoside was produced was confirmed using TLC in the same manner as in Example 1 (1). The results are shown in Table 1. In Table 1, it shows that there is so much production amount that there are many +.

[Example 4] Purification of gingerol glycoside Equal amounts of ethyl acetate were added to the reaction solution (20 mL) obtained in Example 1 to extract the produced glycoside and unreacted 6-gingerol, and unreacted maltose. Was removed. After removing the solvent from the ethyl acetate fraction by an evaporator, hexane was added to the dried solid in an amount equal to that of ethyl acetate, and unreacted 6-gingerol was dissolved and removed. The 6-gingerol glycoside solution can be obtained by dissolving the remaining residue in ethyl acetate (5 mL) again.

  The glycosyltransferase derived from the microorganism belonging to the genus Encipher and the culture of the microorganism of the present invention can be used for glycosylation of a compound having an OH group, and in particular, a glycoside of gingerol can be used for production. . The composition containing the produced gingerol glycoside is superior in water solubility and stability as compared to gingerol, and can be used by being blended in foods, drinks, cosmetics, pharmaceuticals and the like.

Claims (13)

A culture of a microorganism belonging to the genus Ensifer for use in producing a glycoside of the compound in the presence of a compound having an OH group and a sugar donor, the compound having an OH group Is a compound having an alcoholic OH group, and a microorganism belonging to the genus Encipher is a microorganism belonging to the genus Encipher having an activity to produce a glycoside of 6-gingerol in the presence of 6-gingerol and maltose, and sugar donation A culture of a microorganism belonging to the genus Encipher, wherein the body is a sugar donor capable of becoming a glycosyl group or maltosyl group constituting a glycoside . The microorganisms belonging to the genus Encipher are E. adhaerens, E. arboris, E. fredii, E. garamanticus, Encipher costiensis ( E.kostiensis), Encipher Medicae, E.meliloti, E.saheli, E.terangae, E.xinjiangensis And microorganisms selected from the group consisting of S. americanum (S. americannum). The microorganism culture according to claim 2 , wherein the microorganism belonging to the genus Encipher is Encipher adherence. The microorganism culture according to any one of claims 1 to 3, wherein the compound having an OH group is gingerol. The compound or extract containing a compound having an OH group with OH groups, coordination compounds with step a including O H groups exerting Ensifer (Ensifer) culture of a microorganism belonging to the genus in the presence of a sugar donor A method for producing a saccharide, wherein the compound having an OH group is a compound having an alcoholic OH group, and a microorganism belonging to the genus Encipher produces a glycoside of 6-gingerol in the presence of 6-gingerol and maltose A method for producing a glycoside, wherein the sugar donor is a sugar donor that can be a glycosyl group or a maltosyl group constituting the glycoside . The microorganisms belonging to the genus Encipher are Encipher Adherence, Encipher Arboris, Encipher Freddy, Encipher Galamanticus, Ensipher Costiensis, Ensipher Medica, Encipher Meriloti, Encipher Saheli, Encipher Teranga, Encipher Kinzian The method for producing a glycoside according to claim 5, wherein the microorganism is any microorganism selected from the group consisting of Ensis and Synorizobium americanum. The method for producing a glycoside according to claim 6, wherein the microorganism belonging to the genus Encipher is Encipher adherence. The production of a glycoside according to any one of claims 5 to 7, wherein the compound having an OH group and the extract containing the compound having an OH group are gingerol and a plant extract containing gingerol, respectively. Method. Glycoside compounds with OH groups, according to claim 5 to 8 glucose OH group of the compound, maltose or maltooligosaccharides is characterized in that it is a glycoside compound having a dehydration condensation alcoholic OH group The manufacturing method of the glycoside in any one of. The method for producing a glycoside according to any one of claims 5 to 9, wherein the glycoside is a gingerol glycoside represented by the following general formula (1).

(In the formula, R represents a glucosyl group, a maltosyl group or a glucosyl group of maltooligosaccharide, and n represents an integer of 2, 4, 6 or 8.) The gingerol glycoside represented by the general formula (1) is a gingerol glycoside in which glucose is bound to the OH group of the side chain of 6-gingerol represented by the following formula (2) at the α-1 position. The method for producing a glycoside according to claim 10.
The gingerol glycoside represented by the general formula (1) is a gingerol glycoside represented by the following formula (3) in which maltose is bonded to the OH group of the side chain of 6-gingerol at the α-1 position. The method for producing a glycoside according to claim 10.
The method for producing a glycoside according to any one of claims 8 to 12, wherein the plant is ginger.