Methods for producing sesaminol or sesaminol glycosides

Abstract

To provide production methods to reduce production cost of sesaminol or sesaminol glycosides.SOLUTION: Disclosed is a method for producing sesaminol or sesaminol glycosides comprising a step of: reacting a protein with a substrate sesaminol glycoside having at least one glycoside linkage for catalyzing hydrolysis of the at least one glycoside linkage, where the protein is selected from the group consisting of: (1) a protein composed of a specific amino acid sequence; (2) a protein composed of an amino acid sequence having deletion, substitution, insertion and / or addition of one or more amino acids in the specific amino acid sequence and having the activity to catalyze hydrolysis of at least one glycoside linkage; (3) a protein composed of an amino acid sequence with 60% or more sequence identity to the specific amino acid sequence and having the activity to catalyze hydrolysis of at least one glycoside linkage.SELECTED DRAWING: Figure 8

Description

Technical Field

[0001] The present invention relates to a method for producing a compound, and particularly to a method for producing sesaminol or sesaminol glycoside, but the present invention is not limited thereto.

Background Art

[0002] Sesamum indicum L is a plant of the genus Sesamum in the Pedaliaceae family, with a long history of cultivation. In the early days, its main uses were as food and medicine. According to research, sesame contains lignans as bioactive components, and lignans are also contained in sesame processing by-products.

[0003] Lignans are a class of plant secondary metabolites, a type of plant secondary metabolite formed by the oxidative coupling of two molecules of p-hydroxyphenyl propane. They can be divided into two types according to water solubility. Oil-soluble lignans mainly include sesamin, sesamolin, sesamol, sesaminol, sesamolinol, pinoresinol, etc. contained in sesame oil. In addition, lignans exist in the form of glycosides, and lignan glucosides include sesaminol glucosides (SGs), pinoresinol glucosides, etc.

[0004] In recent years, it has been found that sesaminol has stronger antioxidant properties than other sesame lignans and can reduce the onset of Alzheimer's disease and prevent Parkinson's disease. However, the content of sesaminol in sesame is low and it mainly exists in the form of glycosides. Therefore, efficiently hydrolyzing sesaminol glycoside to obtain sesaminol has become an important issue in this field.

[0005] A common method for producing sesaminol is to hydrolyze defatted sesame meal with commercially available enzymes. However, this production process is time-consuming, requires the addition of a large amount of enzymes, and is disadvantageous in terms of cost from a commercial perspective.

Summary of the Invention

Problems to be Solved by the Invention

[0006] In the prior art, the method for producing sesaminol is generally to hydrolyze defatted sesame meal with commercially available enzymes. However, this production process is time-consuming, requires the addition of a large amount of enzymes, and is disadvantageous in terms of cost from a commercial perspective. Therefore, an object of the present invention is to provide a method that can efficiently produce a large amount of sesaminol, shorten the process time, reduce costs, and improve commercial benefits. The inventors of the present application have found that the glycosidic bond of sesaminol glycoside can be hydrolyzed using the enzyme Kmbgl1 to produce sesaminol, and based on this, a method for efficiently producing sesaminol using crude sesame meal extract as a raw material has been established.

Means for Solving the Problems

[0007] Specifically, one aspect of the present invention provides a method for producing sesaminol or sesaminol glycoside, which includes a step of reacting a protein selected from the group consisting of the following (1) to (3) with a substrate sesaminol glycoside having at least one glycosidic bond to catalyze the hydrolysis of the at least one glycosidic bond. (1) A protein consisting of the amino acid sequence of SEQ ID NO: 1; (2) A protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 1 and having an activity of catalyzing the hydrolysis of the at least one glycosidic bond; (3) A protein consisting of an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 and having an activity of catalyzing the hydrolysis of the at least one glycosidic bond.

[0008] According to one embodiment of the present invention, the substrate sesaminol glycoside is selected from the group consisting of sesaminol 2'-O-β-D-glucopyranoside (sesaminol monoglucoside, SMG), sesaminol 2'-O-β-D-glucopyranosyl(1-2)-O-β-D-glucopyranoside (sesaminol (1-2) diglucoside, SDG(1,2)), sesaminol 2'-O-β-D-glucopyranosyl(1-6)-O-β-D-glucopyranoside (sesaminol (1-6) diglucoside, SDG(1,6)), and sesaminol 2'-O-β-D-glucopyranosyl(1-2)-O-(-β-D-glucopyranosyl(1-2))β-D-glucopyranoside (sesaminol triglucoside, STG).

[0009] According to one embodiment of the present invention, the substrate sesaminol glycoside is a -60% (v / v) methanol crude extract.

[0010] According to one embodiment of the present invention, the sesaminol or sesaminol glycoside is selected from the group consisting of sesaminol (1-6) diglucoside (SDG(1,6)), sesaminol (1-2) diglucoside (SDG(1,2)), and sesaminol.

[0011] According to one embodiment of the present invention, the temperature at which the protein reacts with the substrate sesaminol glycoside is in the range of 37 to 45°C.

[0012] According to one embodiment of the present invention, the reaction between the protein and the substrate sesaminol glycoside is carried out under the conditions of pH 5.5 to 6.5.

[0013] According to one embodiment of the present invention, the reaction time between the protein and the substrate sesaminol glycoside is 16 hours.

[0014] According to one embodiment of the present invention, the at least one glycosidic bond is selected from the group consisting of a glycosidic bond between glucose bonded to the 2'-position of sesaminol and an aglycone, a β-1,6-glycosidic bond of gentiobiose bonded to the 2'-position of sesaminol, a β-1,2 bond of sophorose bonded to the 2'-position of sesaminol, a β-1,6-glycosidic bond and a β-1,2 bond of a branched trisaccharide bonded to the 2'-position of sesaminol.

[0015] Another aspect of the present invention provides a method for producing sesaminol or a sesaminol glycoside, which comprises reacting an enzyme derived from a non-human transformed cell into which a polynucleotide selected from the group consisting of the following (1) to (5) has been introduced in a host cell with a substrate sesaminol glycoside having at least one glycosidic bond to catalyze hydrolysis of the at least one glycosidic bond. (1) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1; (2) A polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having an activity of catalyzing hydrolysis of the at least one glycosidic bond; (3) A polynucleotide encoding a protein consisting of an amino acid sequence having 60% or more sequence identity to the amino acid sequence of SEQ ID NO: 1 and having an activity of catalyzing hydrolysis of the at least one glycosidic bond; (4) A polynucleotide that hybridizes under highly stringent conditions with a polynucleotide consisting of a complementary base sequence to the polynucleotide of the protein consisting of the amino acid sequence of SEQ ID NO: 1 and encodes a protein having an activity of catalyzing hydrolysis of the at least one glycosidic bond; (5) A polynucleotide consisting of the base sequence of SEQ ID NO: 2.

[0016] According to one embodiment of the present invention, the polynucleotide is inserted into an expression vector.

[0017] According to one embodiment of the present invention, the non-human transformed cell is selected from the group consisting of transformed plant cells, transformed animal cells, transformed insect cells, transformed Escherichia coli, transformed Bacillus subtilis, transformed Actinomycetes, transformed bacteria, transformed yeast, and transformed filamentous fungi.

[0018] According to one embodiment of the present invention, the substrate sesaminol glycoside is selected from the group consisting of sesaminol 2′-O-β-D-glucopyranoside (sesaminol monoglucoside, SMG), sesaminol 2′-O-β-D-glucopyranosyl(1-2)-O-β-D-glucopyranoside (sesaminol (1-2) diglucoside, SDG(1,2)), sesaminol 2′-O-β-D-glucopyranosyl(1-6)-O-β-D-glucopyranoside (sesaminol (1-6) diglucoside, SDG(1,6)), and sesaminol 2′-O-β-D-glucopyranosyl(1-2)-O-(-β-D-glucopyranosyl(1-2))β-D-glucopyranoside (sesaminol triglucoside, STG).

[0019] According to one embodiment of the present invention, the sesaminol or sesaminol glycoside is selected from the group consisting of sesaminol (1-6) diglucoside (SDG(1,6)), sesaminol (1-2) diglucoside (SDG(1,2)), and sesaminol.

[0020] According to one embodiment of the present invention, the at least one glycosidic bond is selected from the group consisting of a glycosidic bond between glucose bonded to the 2′-position of sesaminol and an aglycone, a β-1,6-glycosidic bond of gentiobiose bonded to the 2′-position of sesaminol, a β-1,2 bond of sophorose bonded to the 2′-position of sesaminol, a β-1,6-glycosidic bond and a β-1,2 bond of a branched trisaccharide bonded to the 2′-position of sesaminol.

Advantages of the Invention

[0021] As an advantage of the method provided by the present invention, not only can the manufacturing process time be shortened, but also the production amount of sesaminol can be increased to reduce the production cost, thus meeting the requirements of industrial applicability.

[0022] Hereinafter, the technology of the present invention will be described in detail with reference to the accompanying drawings and examples.

Brief Description of the Drawings

[0023]

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Embodiments for Carrying Out the Invention

[0024] It should be understood that the various aspects are not limited to the arrangements and means shown in the drawings.

[0025] The following embodiments are not intended to limit the present invention only to these embodiments. Those skilled in the art can make modifications and changes to the embodiments discussed herein without departing from the spirit or scope of the present invention, and they are included within the scope of the present invention.

[0026] As used herein, the terms "comprising", "including", "having" or "containing" have an open-ended and non-limiting meaning unless the context clearly indicates otherwise, and do not exclude other unstated elements or method steps. The terms "a" and "the" may be intended to include the plural as well, unless the context clearly indicates that they are singular. The term "one or more" means "at least one", and thus can include a single feature or a mixture / combination.

[0027] The numerical values or experimental data provided in this specification are basically approximate numerical values, and the relevant numerical values in specific embodiments are presented here as accurately as possible. However, each numerical value inherently includes the standard deviation resulting from the individual test methods used. Here, the term "about" is used with the intention that the actual value falls within the allowable range of the average standard error determined by those skilled in the art.

[0028] The present invention provides a method for producing sesaminol or sesaminol glucoside, which includes reacting a protein with a substrate sesaminol glucoside having at least one glycosidic bond to catalyze the hydrolysis of the at least one glycosidic bond. In this specification, "sesaminol glucosides (SGs)" means a glycoside in which sesaminol and a sugar are bonded, and is also referred to as "sesaminol glucoside".

[0029] According to one embodiment of the present invention, the protein is selected from the group consisting of the following (1) to (3). (1) A protein consisting of the amino acid sequence of SEQ ID NO: 1 (MSKFDVEQLLSELNQDEKISLLSAVDFWHTKKIERLGIPAVRVSDGPNGIRGTKFFDGVPSGCFPNGTGLASTFDRDLLETAGKLMAKESIAKNAAVILGPTTNMQRGPLGGRGFESFSEDPYLAGMATSSVVKGMQGEGIAATVKHFVCNDLEDQRFSSNSIVSERALREIYLEPFRLAVKHANPVCIMTAYNKVNGEHCSQSKKLLIDILRDEWKWDGMLMSDWFGTYTTAAAIKNGLDIEFPGPTRWRTRALVSHSLNSREQITTEDVDDRVRQVLKMIKFVVDNLEKTGIVENGPESTSNNTKETSDLLRKIAADSIVLLKNKNNILPLKKEDNIIVIGPNAKAKTSSGGGSASMNSYYVVSPYEGIVNKLGKEVDYTVGAYSHKSIGGLAESSLIDAAKPADAENSGLIAKFYSNPVEERSDDEEPFHVTKVNRSNVHLFDFKHEKVDPKNPYFFVTLTGQYVPQEDGDYIFSLQVYGSGLFYLNDELIIDQKHNQERGSFCFGAGTKERTKKLTLKKGQVYNVRVEYGSGPTSGLVGEFGAGGFQAGVIKAIDDDEEIRNAAELAAKHDKAVLIIGLNGEWETEGYDRENMDLPKRTNELVRAVLKANPNTVIVNQSGTPVEFPWLEDANALVQAWYGGNELGNAIADVLYGDVVPNGKLSLSWPFKLQDNPAFLNFKTEFGRVIYGEDIFVGYRYYEKLQRKVAFPFGYGLSYTTFELDISDFKVTDDKIAISVDVKNTGDKFAGSEVVQVYFSALNSKVSRPVKELKGFEKVHLEPGEKKTVNIDLELKDAISYFNEELGKWHVEAGEYLVSVGTSSDDILSVKEFKVEKELYWKGL), (2) SEQ IDA protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of NO:1), and having an activity of catalyzing the hydrolysis of said at least one glycosidic bond, (3) a protein consisting of an amino acid sequence having a sequence identity of 60% or more with respect to the amino acid sequence of SEQ ID NO:1, and having an activity of catalyzing the hydrolysis of said at least one glycosidic bond.

[0030] Said SEQ ID NO:1 is derived from Kluyveromyces marxianus. Kluyveromyces marxianus belongs to the kingdom Fungi and is a kind of yeast. According to at least one embodiment of the present invention, the protein consisting of the amino acid sequence of SEQ ID NO:1 is derived from the yeast enzyme Kmbgl1. Specifically, the structure is composed of four parts: (α / β)8 barrel-like (corresponding to positions 1 to 295 of SEQ ID NO:1), PA14 domain (positions 392 to 559), (α / β)6 sandwich (positions 307 to 381, 560 to 658) and C-terminal domain (positions 700 to 845). It belongs to the glycosidase hydrolase family 3 (GH3) and can hydrolyze sesaminol triglucoside (STG) or sesaminol diglucoside (SDG) to produce sesaminol and sesaminol glycosides. The active site of Kmbgl1 contains D225 and E590, and also contains a plurality of glucose binding sites, such as D45, L99, R113, K146, H147, R157, M190, Y193, D225, W266, S356, F445, F508, E590, etc.

[0031] According to at least one embodiment of the present invention, the enzyme of the present invention uses a protein consisting of the amino acid sequence of SEQ ID NO: 1 or a variant thereof, and the variant includes those artificially obtained. Further, the "amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added" includes, for example, 1 to 3, 1 to 5, 1 to 10, 1 to 20, 1 to 50, 1 to 80 amino acids being deleted, substituted, inserted and / or added. Generally, the smaller the number of deletions, substitutions, insertions and / or additions of the amino acids, the more preferable. Further, the amino acid sequence having 60% or more sequence identity includes, for example, but is not limited to, an amino acid sequence having 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 97% or more, 98% or more or 99% or more sequence identity. Preferably, the amino acid sequence has 75% or more sequence identity.

[0032] In the present invention, the "substrate sesaminol glycoside" means a sesaminol glycoside that serves as a substrate for the reaction. According to one embodiment of the present invention, the substrate sesaminol glycoside is selected from the group consisting of sesaminol 2'-O-β-D-glucopyranoside (sesaminol monoglucoside, SMG), sesaminol 2'-O-β-D-glucopyranosyl(1-2)-O-β-D-glucopyranoside (sesaminol (1-2) diglucoside, SDG(1,2)), sesaminol 2'-O-β-D-glucopyranosyl(1-6)-O-β-D-glucopyranoside (sesaminol (1-6) diglucoside, SDG(1,6)) and sesaminol 2'-O-β-D-glucopyranosyl(1-2)-O-(-β-D-glucopyranosyl(1-2))β-D-glucopyranoside (sesaminol triglucoside, STG). Preferably, the substrate sesaminol glycoside is sesaminol triglucoside (STG). According to one embodiment of the present invention, the rare sesaminol glycosides such as SDG(1,2) or SDG(1,6) can be generated by reacting the most abundant sesaminol glycoside STG in the pressed sesame oil cake as a substrate through the enzyme provided by the present invention.

[0033] In this specification, the "at least one glycosidic bond of sesaminol glycoside" means, in sesaminol glycoside, the glycosidic bond (also referred to as glycosidic linkage) between sesaminol and the side chain, and the glycosidic bonds within the side chain. Further, the "activity of hydrolyzing the glycosidic bond of a substrate sesaminol glycoside having at least one glycosidic bond" means the activity of hydrolyzing (cleaving) at least one glycosidic bond in the sesaminol glycoside. According to one embodiment of the present invention, the at least one glycosidic bond is selected from the group consisting of a glycosidic bond between glucose bonded to the 2'-position of sesaminol and an aglycone, a β-1,6-glycosidic bond of gentiobiose bonded to the 2'-position of sesaminol, a β-1,2 bond of sophorose bonded to the 2'-position of sesaminol, a β-1,6-glycosidic bond and a β-1,2 bond of a branched trisaccharide bonded to the 2'-position of sesaminol. According to one embodiment of the present invention, in the hydrolysis of the glycosidic bond, all glycosidic bonds are hydrolyzed to produce sesaminol from the sesaminol glycoside. In another embodiment, the β-1,6-glycosidic bond of the branched trisaccharide bonded to the 2'-position of sesaminol is preferentially hydrolyzed. In this way, by the production method of the present invention, a produced sesaminol glycoside in which only some glycosidic bonds are cleaved or sesaminol in which all glycosidic bonds are cleaved can be produced.

[0034] It should be noted that the inventors of the present application have found that at least one of the amount of protein added, the substrate extraction form, the reaction temperature, the reaction pH value, and the reaction time affects the production status of sesaminol or sesaminol glycoside.

[0035] According to a preferred embodiment of the present invention, the substrate sesaminol glycoside is a -60% (v / v) methanol crude extract. Specifically, the 60% (v / v) methanol crude extract is obtained by extracting sesame meal at room temperature using 60% (v / v) methanol.

[0036] According to a preferred embodiment of the present invention, the temperature at which the protein reacts with the substrate sesaminol glycoside is in the range of 37 to 45 °C, and is a value within the range between any one or any two of the following values, for example, 37 °C, 38 °C, 39 °C, 40 °C, 41 °C, 42 °C, 43 °C, 44 °C, and 45 °C, but is not limited thereto. More preferably, the temperature at which the protein reacts with the substrate sesaminol glycoside is 40 °C.

[0037] According to a preferred embodiment of the present invention, the reaction between the protein and the substrate sesaminol glycoside is carried out under the conditions of pH 5.5 to 6.5, and is a value within the range between any one or any two of the following values, for example, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, and pH 6.5, but is not limited thereto. More preferably, the reaction between the protein and the substrate sesaminol glycoside is carried out under the condition of pH 6.5.

[0038] According to a preferred embodiment of the present invention, the reaction time between the protein and the substrate sesaminol glycoside is in the range of 12 to 16 hours, and is a value within the range between any one or any two of the following values, for example, 12 hours, 13 hours, 14 hours, 15 hours, and 16 hours, but is not limited thereto. More preferably, the reaction time between the protein and the substrate sesaminol glycoside is 16 hours.

[0039] Another aspect of the present invention provides a method for producing sesaminol or sesaminol glycoside, which comprises reacting an enzyme derived from a non-human transformed cell with a substrate sesaminol glycoside having at least one glycosidic bond in a host cell to catalyze the hydrolysis of the at least one glycosidic bond.

[0040]

[0041] According to at least one embodiment of the present invention, the polynucleotide sequence encoding the protein consisting of the amino acid sequence of SEQ ID NO: 1 is a polynucleotide encoding the enzyme KmBgl1 of yeast Kluyveromyces marxianus, and more specifically, it is a DNA sequence that can be transcribed and translated to produce the enzyme KmBgl1.

[0042] According to one embodiment of the present invention, the non-human transformed cell is selected from the group consisting of transformed plant cells, transformed animal cells, transformed insect cells, transformed Escherichia coli, transformed Bacillus subtilis, transformed actinomycetes, transformed bacteria, transformed yeast, and transformed filamentous fungi.

[0043] According to one embodiment of the present invention, the substrate sesaminol glycoside is selected from the group consisting of sesaminol 2′-O-β-D-glucopyranoside (sesaminol monoglucoside, SMG), sesaminol 2′-O-β-D-glucopyranosyl(1-2)-O-β-D-glucopyranoside (sesaminol(1-2)diglucoside, SDG(1,2)), sesaminol 2′-O-β-D-glucopyranosyl(1-6)-O-β-D-glucopyranoside (sesaminol(1-6)diglucoside, SDG(1,6)), and sesaminol 2′-O-β-D-glucopyranosyl(1-2)-O-(-β-D-glucopyranosyl(1-2))β-D-glucopyranoside (sesaminol triglucoside, STG).

[0044] According to one embodiment of the present invention, the at least one glycosidic bond is selected from the group consisting of a glycosidic bond between glucose bound to the 2′-position of sesaminol and an aglycone, a β-1,6-glycosidic bond of gentiobiose bound to the 2′-position of sesaminol, a β-1,2 bond of sophorose bound to the 2′-position of sesaminol, a β-1,6-glycosidic bond and a β-1,2 bond of a branched trisaccharide bound to the 2′-position of sesaminol.

[0045] In this specification, "polynucleotide" means DNA or RNA. According to one embodiment of the present invention, the polynucleotide is inserted into an expression vector. Specifically, the polynucleotide of the present invention is preferably introduced into a host in a state where it is inserted into an appropriate expression vector. An appropriate expression vector is usually constructed to include the following (1) to (3). (1) A promoter that can be transcribed in a host cell, (2) the polynucleotide of the present invention bound to the promoter, and (3) an expression cassette comprising a signal capable of exerting functions related to transcription termination and polyadenylation of RNA molecules in the host cell as a constituent element. Examples of methods for preparing an expression vector include methods using plasmids, phages, or cosmids, but the present invention is not particularly limited. Also, the specific type of vector in the present invention is not particularly limited, and a vector capable of being expressed in a host cell may be appropriately selected. That is, according to the type of host cell, an appropriate promoter sequence is selected as appropriate to ensure the expression of the polynucleotide of the present invention, and a vector in which this and the polynucleotide of the present invention are incorporated into various plasmids or the like may be used as an expression vector.

[0046] According to a preferred embodiment of the present invention, the polynucleotide of the present invention may further include a polynucleotide consisting of a nucleotide sequence encoding a secretion signal peptide. More preferably, the polynucleotide consisting of the nucleotide sequence encoding the secretion signal peptide is included at the 5'-end of the polynucleotide of the present invention.

[0047] The expression vector of the present invention depends on the type of host to be introduced and contains expression control regions (such as promoters, terminators, and / or origins of replication, etc.). As the promoter of the expression vector for bacteria, conventional promoters (such as trc promoter, tac promoter, lac promoter, etc.) can be used. As the promoter for yeast, for example, glyceraldehyde 3-phosphate dehydrogenase promoter, PH05 promoter, etc. can be mentioned. As the promoter for filamentous fungi, for example, amylase, trpC, etc. can be mentioned. Also, examples of promoters for expressing a target gene in plant cells include cauliflower mosaic virus 35S RNA promoter, rd29A gene promoter, rbcS promoter, and mac-1 promoter in which the enhancer sequence of the 35S RNA promoter of the cauliflower mosaic virus is added to the 5'-side of the mannopine synthase promoter sequence derived from Agrobacterium. As the promoter for animal cell hosts, viral promoters (such as SV40 early promoter, SV40 late promoter, etc.) can be mentioned. Examples of promoters that are inductively activated by external stimuli include mouse mammary tumor virus (MMTV) promoter, tetracycline-responsive promoter, metallothionein promoter, and heat shock protein promoter, etc.

[0048] The expression vector preferably contains at least one selectable marker. Such markers include auxotrophic markers (ura5, niaD), drug resistance markers (hygromycin, Zeocin), geneticin resistance gene (G418r), copper resistance gene (CUP1), celenin resistance genes (fas2m, PDR4), etc.

[0049] The method for producing the transformant of the present invention is not particularly limited. For example, a method of introducing an expression vector containing the polynucleotide of the present invention into a host and transforming it can be mentioned. As the cells or organisms to be transformed, various conventionally known cells or organisms can be appropriately used. As the cells to be transformed, for example, bacteria such as Escherichia coli, yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe), filamentous fungi (Aspergillus oryzae, Aspergillus sojae), plant cells, animal cells excluding humans, etc. can be mentioned. Appropriate media and conditions used for the above host cells are well-known in the art. Also, the organisms to be transformed are not particularly limited, and various microorganisms, plants, or animals excluding humans exemplified by the above host cells can be mentioned. The transformant is preferably a filamentous fungus, yeast, or plant. As the host used in the transformation, one that can produce any of the sesaminol glycosides can also be used. Not only plants such as sesame that can originally produce at least one sesaminol glycoside, but also those obtained by introducing genes necessary for the production of at least one sesaminol glycoside into cells or organisms that do not originally produce sesaminol glycosides can be used as the host.

[0050] As the method for transforming the host cell, generally used well-known methods can be utilized. For example, it can be carried out by the methods described in the electroporation method, particle delivery method, spheroplast method, or lithium acetate method, etc., but is not limited thereto. Also, when introducing a gene into a plant, or a tissue or cell derived from a plant, for example, the Agrobacterium method, particle gun method, PEG method, or electroporation method, etc. can be appropriately selected and used.

[0051] The protein of the present invention can be obtained by expressing the protein in a host cell and disrupting the cell. By reacting the protein of the present invention with a substrate sesaminol glycoside, the sesaminol glycoside and / or sesaminol of the present invention can also be produced.

[0052] The "enzyme derived from the transformed cell" described above is prepared using the transformed cell and is not particularly limited as long as it contains the protein of the present invention. For example, it can be the transformed cell itself, the disrupted product of the transformed cell itself, the culture supernatant of the transformed cell itself, and purified products thereof. Therefore, the present invention provides a method for producing sesaminol and / or sesaminol glycoside, which comprises a step of contacting an enzyme derived from a non-human transformed cell with a sesaminol glycoside having at least one glycosidic bond in a host cell to hydrolyze at least one glycosidic bond.

[0053] The "contact" described above means allowing the enzyme derived from the transformed cell of the present invention and the sesaminol glycoside having at least one glycosidic bond to be present in the same reaction system or culture system. For example, adding a sesaminol glycoside having at least one glycosidic bond to a container containing the enzyme derived from the transformed cell of the present invention, mixing a sesaminol glycoside having at least one glycosidic bond with a sesaminol glycoside having at least one glycosidic bond, or adding a sesaminol glycoside having at least one glycosidic bond to a container containing a sesaminol glycoside having at least one glycosidic bond.

[0054] When the transformant is yeast or Aspergillus, the yeast or Aspergillus transformed with the polynucleotide of the present invention expresses more of the protein of the present invention than the wild type. Furthermore, the expressed protein reacts with the sesaminol glycoside produced in the yeast or Aspergillus, and sesaminol and / or sesaminol glycoside is produced in the cells or culture broth of the yeast or Aspergillus, preferably in the culture broth.

[0055] When the transformant is a plant, the plant to be transformed in the present invention means the whole plant body, plant organs (such as leaves, petals, stems, roots, seeds, etc.), plant tissues (such as epidermis, phloem, parenchyma, xylem, vascular bundle, palisade tissue, spongy tissue, etc.) or plant cultured cells, or various forms of plant cells (such as suspension-cultured cells), protoplasts, leaf sections, callus, etc. The plant used for transformation may be any plant belonging to the class Monocotyledoneae or Dicotyledoneae. Whether the polynucleotide of the present invention has been introduced into a plant can be confirmed by methods such as PCR method, Southern hybridization method, Northern hybridization method, etc. Once a transformed plant in which the polynucleotide of the present invention has been integrated into the genome is obtained, progeny can be obtained by sexual reproduction or asexual reproduction of the plant. Further, from the plant or its progeny, or clones thereof, for example, seeds, fruits, cuttings, tubers, tuberous roots, strains, callus, protoplasts, etc. can be obtained, and based on these, the plant can be mass-produced. The plant transformed with the polynucleotide of the present invention (hereinafter referred to as "the plant of the present invention") contains more of the protein of the present invention than the wild type. Therefore, the protein of the present invention reacts with the sesaminol glycoside produced in the plant, and sesaminol is produced in the plant. However, when the environment in the plant body is not optimal for the hydrolysis reaction, the hydrolysis reaction of the glycoside bond of the sesaminol glycoside is suppressed, and the sesaminol glycoside with the glycoside bond maintained without cleavage or the sesaminol glycoside with only a part of the glycoside bond cleaved is produced.

[0056] In some embodiments of the present invention, the transformant or its culture solution has a higher content of sesaminol and / or sesaminol glycoside compared to its wild type, and its extract or culture solution contains a high concentration of sesaminol and / or sesaminol glycoside. The extract of the transformant of the present invention can be obtained by crushing the transformant with glass beads, a homogenizer or a sonicator, centrifuging the crushed product, and recovering the supernatant. When the sesaminol and / or sesaminol glycoside of the present invention accumulates in the culture solution, after the culture is completed, the transformant and the culture supernatant are separated by a normal method (such as centrifugation, filtration, etc.), and the culture supernatant containing the sesaminol and / or sesaminol glycoside of the present invention can be obtained.

Examples

[0057] Hereinafter, the present invention will be described in detail with reference to examples. However, it should be understood that these examples are intended to assist in the understanding of the present invention and are not intended to limit the scope of the present invention in any way.

[0058] A. Experimental method Extraction and analysis of sesame meal samples Sample treatment: The sesame meal is ground with a mill, passed through a 20-mesh sieve, 10 g of the powder is taken, 100 mL of n-hexane is added, stirred overnight at room temperature, the filtrate containing residual oil is suction filtered, the solid is filtered off, and the defatted by-product powder is obtained by drying in an oven at 40 °C.

[0059] Analysis method: Weigh 1 g of defatted sesame meal, add 20 mL of 60% methanol, perform ultrasonic vibration extraction for 1 hour, centrifuge (9000 rpm, 10 minutes), take the supernatant, add 20 mL of 60% methanol to the precipitate, perform ultrasonic vibration extraction for 1 hour, centrifuge (9000 rpm, 10 minutes), combine all the supernatants, make up to 50 mL in a volumetric flask, dilute appropriately, and analyze by HPLC.

[0060] Preparation of sesame meal extract 1. 60% (v / v) methanol extract 150 g of defatted sesame meal was taken, stirred and extracted overnight at room temperature with 10 times the amount of 60% methanol, the filtrate was suction filtered, concentrated under reduced pressure to remove the organic solvent, and freeze-dried to obtain a powder.

[0061] 2. Hot water extract 150 g of sesame meal was taken, extracted with 10 times the amount of water at 121 °C for 1 hour, centrifuged (9000 rpm, 10 minutes) after cooling, the supernatant was taken, and freeze-dried to obtain a powder.

[0062] 3. 50% (v / v) and 60% (v / v) ethanol extracts 150 g of defatted sesame meal was taken, stirred and extracted overnight at room temperature with 10 times the amount of 50% and 60% ethanol respectively, the filtrate was suction filtered, concentrated under reduced pressure to remove the organic solvent, placed in a vacuum oven at 40 °C and dried overnight, the weight of the extract was weighed, and the solid content was calculated.

[0063] Enzymatic hydrolysis reaction 1 g of the 60% methanol extract was suspended in 3 mL of 50 mM pH 5.5 citrate phosphate buffer (STG content was 1.6 mg / mL), 114 U (4 mg / mL) of Kmbgl1 was added, reacted at 45 °C for 30 minutes, an equal amount of methanol was added to stop the reaction, centrifuged (1500 rpm, 10 minutes), the supernatant was taken, appropriately diluted, and analyzed by HPLC.

[0064] Expression and separation and purification of Kmbgl1 enzyme 1. Activation of the strain and inoculum culture The Kmbgl1::pET21a(+) / E. coli frozen stock (-80 °C) strain was inoculated onto a Lysogeny broth (LB) solid medium plate containing 100 μg / mL ampicillin, cultured at 37 °C for 16 hours, a single colony was taken out, inoculated into 100 mL of LB liquid medium containing 100 μg / mL ampicillin, and cultured at 37 °C, 160 rpm for 16 hours, and used as the inoculum.

[0065] 2. Induction of Mass Expression of Recombinant Protein Gene by IPTG Inoculate 5 mL of the inoculum into 500 mL of TB liquid medium containing 100 μg / mL ampicillin, and culture at 37 °C and 150 rpm until OD 600 = 0.6. Next, add 200 μL of 500 mM IPTG, and induce the expression of the recombinant protein at 16 °C and 110 rpm for 24 hours at low temperature. Centrifuge the bacterial cells (6000 rpm, 10 minutes, 4 °C), collect the bacterial cell precipitate, and store it at -20 °C.

[0066] Resuspend the bacterial cells of 500 mL of the bacterial solution in 50 mL of cell lysis buffer, shake until there are no bacterial clumps in the solution, add 250 μL of 200 mM protease inhibitor (PMSF), disrupt the bacteria by sonication for 30 minutes (disrupt the bacteria for 8 seconds and let it stand for 4 seconds), remove the cell debris by low-temperature centrifugation (15000 rpm, 4 °C, 30 minutes), collect the supernatant to obtain a crude enzyme extract, rapidly freeze it in liquid nitrogen after purification, and store it at -80 °C.

[0067] Analysis of Kmbgl1 Affinity The Kmbgl1 sequence was loaded into BLAST software for phylogenetic comparative analysis, and the results are shown in Table 1 below. In order to test the hydrolysis ability (not shown in the figure) of these species, the inventors of the present application carried out an enzymatic hydrolysis reaction of the species in this analysis result. The results showed that the species Kluyveromyces lactis, Lachancea fermentati, Saccharomyces mikatae IFO 1815, Brettanomyces anomalus, and Clavispora sp. NRRL Y-50464 all had a sequence identity of 55% or more with the enzyme of the present invention. Kluyveromyces lactis and Lachancea fermentati have sesaminol glycoside bond hydrolysis activity and can produce sesaminol. Saccharomyces mikatae IFO 1815, Brettanomyces anomalus, and Clavispora sp. NRRL Y-50464 could not completely hydrolyze sesaminol glycoside into sesaminol, and it was shown that mainly STG and SDG were present after the reaction.

[0068]

Table 1-1

Table 1-2

[0069] Furthermore, the identities of the above species with hydrolysis ability were compared and analyzed, and the results are shown in Table 2.

[0070]

Table 2

[0071] Measurement of the activity of the crude enzyme solution The activity was measured using the absorbance at 405 nm of the yellow p-nitrophenol (PNP) product formed after hydrolysis of the colorless p-nitrophenyl β-D-glucopyranoside (PNPG) glycosidic bond. The activity measurement was carried out before the enzyme experiment for each batch. The substrates for the reaction were 110 μL of citrate-phosphate buffer (50 mM, pH 6.5) and 5 μL of PNPG. After diluting the enzyme to a protein concentration of 6 - 22 ppm, 5 μL of the test enzyme was added, and the reaction was carried out in a dry bath at 55 °C for 10 minutes. The reaction was stopped with 120 μL of 0.5 M sodium carbonate to provide alkaline conditions for color development. The absorbance at 405 nm was measured and applied to the calibration curve of PNP to determine the concentration of PNP (C PNP ) in the 240 μL reaction solution. One unit (U) of enzyme activity was defined as the amount of enzyme that produces 1 μM of PNP per minute, and the activity concentration (U / mL) of the enzyme solution could be calculated using the following formula.

[0072]

Number

[0073] RP-HPLC-UV analysis Analysis conditions · Reverse-phase chromatography column: C18 column YMC-Pack ODS-AM (250x4.6 mm, 5C) · Mobile phase: Aqueous phase 0.1% (v / v) formic acid aqueous solution, organic phase acetonitrile (ACN) · Flow rate: 1.0 mL / min · Sample injection volume: 20 mL · Detection wavelength: 290 nm · Gradient elution

[0074]

Table 3

[0075] B. Experimental results Content analysis of sesaminol-related derivatives in sesame meal Effect of different solvents on the extraction of sesaminol glycosides Here, the difference in effects between using 60% methanol and using 50 - 60% ethanol in the preparation of sesaminol glycosides was explored, and the subsequent substrate preparation method was established.

[0076] The results are shown in Table 3 below. Each extraction solvent was extracted at a ratio of 10:1 (v / w). The example using water as the solvent was carried out by autoclaving at 121 °C for 60 minutes. As can be seen from Table 3, the extraction effect of 60% methanol is the best, and the content of sesaminol glycosides in the raw material can be increased to 10.5%. This extraction method is used with a recovery rate of 100%. There was no significant difference in the extraction rates using 50% and 60% ethanol, and the recovery rate remained at about 86% compared with 60% methanol extraction. When extracted by the autoclaving method, the extraction amount was the largest, but the impurity content was relatively high. Only 5.2 grams of sesaminol glycosides were contained per 100 grams of the extract. During the extraction process, the oil and water in the sesame meal were mixed to cause emulsification, so the obtained filtrate was very turbid, and it was difficult to separate the filtrate and residue by the suction filtration method. Solid-liquid separation was carried out by the high-speed centrifugation method to break the emulsified phase. From the perspective of extraction efficiency, in subsequent examples, basically 60% methanol was used to prepare the sesame meal extract and used as the substrate for the subsequent enzymatic hydrolysis reaction.

[0077]

Table 4

[0078] Expression of Kmbgl1 by E. coli recombinant protein Cultured with the culture method of Escherichia coli, 0.5 mM IPTG was added, the expression of β-glucosidase was induced at low temperature, then the cells were disrupted to collect intracellular proteins, and further purified with a Ni-NTA column. Regarding the coordination bond, a high concentration of imidazole was used to compete with the His6-tag on the target protein. As shown in Figure 1, the initial purification was collected with separate columns, and the results of expression and purification were analyzed by SDS-PAGE. When the imidazole concentration in the eluent was 200 mM, the target protein could be eluted, mainly concentrated in the third and fourth columns, and there was an obvious band at the position of 95 kD, which was the Kmbgl1 enzyme. The protein in the crude enzyme solution and the partially purified enzyme solution was quantified by Bradford assay, and the activity was measured using pNP-β-Glu as the substrate. The protein content of the crude enzyme solution was 0.85 mg / mL, and the activity was 60.42 U / mg. The protein content of the enzyme solution after purification was 0.5 mg / mL, and the activity was 92.14 U / mg. In this way, it can be confirmed that all the cultured enzymes have activity. Then, the active Kmbgl1 crude enzyme solution was used to participate in the reactions of other examples.

[0079] Conversion phenomenon of Kmbgl1 protein in sesame extract Here, the situation where Kmbgl1 catalyzes the conversion of sesaminol glycoside to produce sesaminol and the ability to hydrolyze STG are explored. Specifically, 1 g of sesame meal hot water extract was used as the substrate for the reaction, and 100 U of enzyme was added for the reaction. The results are shown in Figure 2. When reacting at 40 °C, Kmbgl1 can gradually hydrolyze the sesaminol glycoside in the sesame meal extract to produce sesaminol, and directly cleave the glycoside bond on STG and SDG to convert them into sesaminol during the reaction process.

[0080] Test on the hydrolysis conditions of Kmbgl1 enzyme Through the above experiments, aspects such as the substrate of the reaction and its extraction form, as well as the enzyme for catalyzing the reaction, can be almost determined. Next, in order to find the optimal conditions for catalyzing hydrolysis with Kmbgl1, different reaction conditions were tested.

[0081] Effect of Optimal Enzyme Addition Amount on the Production of Sesaminol Glycoside First, the present invention explores the optimal enzyme addition amount. Here, an extract rich in sesaminol glycoside was used as the substrate for the reaction. The results are shown in Figure 3. As the enzyme addition amount increased, the production amount of sesaminol gradually increased. When the enzyme amount was added up to 51.8 U, the conversion rate to sesaminol reached 80% when calculated quantitatively.

[0082] Effect of Optimal Reaction Time on the Production of Sesaminol Glycoside According to the results of the previous test, in this part, 51.8 U of the optimal enzyme amount was added to 0.1 g of the substrate, and then the subsequent experiment was carried out to further explore the optimal reaction time. The results are shown in Figure 4. As the reaction time increased, STG and SDG in the crude extract gradually decreased and were converted into sesaminol. The enzyme continued to act until the reaction continued for 16 - 18 hours under the reaction conditions of 37 °C, and gradually no obvious sesaminol was produced. As can be seen from this, the optimal reaction time of the enzyme of the present invention is 16 hours, and the enzyme may lose its activity after 16 hours. Therefore, the reaction time during the subsequent tests was all set to 16 hours.

[0083] Effect of Optimal Reaction pH Value on the Production of Sesaminol Glycoside Since the pH value is also an important factor affecting the enzyme hydrolysis reaction, this test continues to find the optimal pH value during the reaction of sesaminol glycoside. According to the literature, Kmbgl1 is relatively stable at pH 6.0 - 9.0 and shows better reactivity under weakly acidic conditions. Therefore, in this test, pH 5.5, 6.0, 6.5, and 7.0 were selected as the concentrations for the subsequent experiments. The results are shown in Figure 5. Under the conditions of pH 6.0 and 6.5, the production amount of sesaminol was more, indicating that the optimal reaction pH value of Kmbgl1 for sesaminol glycoside should be between 6.0 and 6.5.

[0084] Effect of Optimal Reaction Temperature on the Production of Sesaminol Glycoside After confirming that the optimal pH values for the hydrolysis of sesaminol glycosides by Kmbgl1 are 6.0 and 6.5, considering that Kmbgl1 is relatively stable at pH 6.5, this test measured the effect of different temperatures on the conversion of sesaminol glycosides under the condition of pH 6.5. The results are shown in Figure 6. At the same substrate mass, enzyme addition amount and reaction pH value, when reacted at 40°C for 16 hours, the sesaminol production rate reached a maximum of 80%. Under the condition of 37°C, during the continuous enzyme reaction, the average amount of sesaminol produced at each time point was almost the same, but the total sesaminol conversion rate was the lowest among the three temperatures, indicating that the enzyme activity was relatively low at this temperature. After reacting at 45°C for 2 hours, the amount of sesaminol produced was more than the other two, but after reacting for up to 12 hours, the amount of sesaminol produced gradually decreased and sometimes no sesaminol was produced, showing that increasing the temperature could indeed increase the reaction rate, but at the same time the reaction possible time was shortened. From the perspective of the final total conversion rate, 40°C was the optimal reaction temperature.

[0085] Hydrolysis tests of different enzymes Here, the differences in the preparation of sesaminol glycosides by enzymes with different sequence identities were explored. Specifically, this test hydrolyzed sesame meal hot water extract (HWE) with crude enzyme solutions from different yeasts and further analyzed the content of the generated sesaminol glycosides. The experiment will be briefly described below. 0.5 g of HWE powder was weighed into a capped test tube, citric acid phosphate buffer (pH 6.5, 50 mM) containing 100 enzyme activity units (U) was added, and after thorough mixing, a 0-hour sample was taken. Then it was placed in a shaking water bath at 40°C and reciprocally shaken at 100 rpm for 16 hours to carry out the reaction. Sampling was done again. For sampling, the enzyme reaction was stopped with an equal volume of methanol, then diluted 40-fold with methanol, centrifuged to remove the precipitate, and the supernatant was taken for RP-HPLC-UV analysis.

[0086] The results are shown in Fig. 7. The enzyme Kmbgl1 of the present invention can effectively convert sesame meal into sesaminol glycosides, and Klbg (SEQ ID NO: 3, MSNFDIEQTLSELTRDEKISLLSAVDFWHTKEIERLGIPSVRVSDGPNGIRGTRFFDSVPSGCFPNGTGLASTFDDELLKEAGKLMAKEAVAKNAAVILGPTTNMQRGPLGGRGFESFSEDPYLAGVATSSVVQGMQSEGIAATVKHFVCNDLEDQRFASNSILSERALREIYLEPFRLAIKNADPVCLMTAYNKVNGEHCSQNKKLLLDILRKEWNWDGMIMSDWYGTYTTAASIKNGLDIEFPGPTRWRTNELVSHSLNSKEQISIYDVDDRVRQVLKMIKFVVDNQEKTGIVQNGPETTSNNTKETSELLRKIAADSIVLLKNENSILPLKKEESIVVIGPNAKAKASSGGGSASVNSYYVISPYEGIVKKVGKEVPYTIGAESHKTLSNLIEQLVVDPSKPAEGDNAGATGSFYSEPVEKRAKDESPFHVATFKHSFNLLFDFKHEKIDTTNPIFYITLEGYFTPEEDADYIFGLQVFGTGVLYLDDELLIDQRKGQVSGDFCFGAGTIEKTKTVTLQKGKAYKVRIEYGSGPTSELVSEFGSGALQVGVTKAIDADEEIKKAAKLAAAHDKAILCIGLNAEWESEGHDREDMTLPARTNDLVRAVLEANPNTVIVNQSGTPVEFPWLQKANALVQAWYGGNELGNAIADVLYGDVVPNGKLSLSWPLKLEDNPAYLNFKTEFGRVVYGEDIFIGYRFYEKLQKRVAFPFGYGLSYTEFALSNLQVQINDEVISVSVDVKNTGEKYAGSEVVQVYIAATESSVSRAVKELKGFKKVLLQPGQTETAKIDLVLKDSVSFFDEEVGKWCSEAGQYKVLVGTSSDDIVLSESFDVEKTSYWSGL) and Lfbg (SEQ ID NO: 4,MSKFDIEELIGELTLQEKIALIAAKDFWHTSPVERLGIPSVRVSDGPNGVRGTKFFNSVPSAAFPNGTGLASTFDTELLEEAGQLMAVEAAHKNASVILGPTTNIARGPLGGRGFESFSEDPYLSGMCTAALVNGMQSRGIAATVKHYVCNDLEDQRFSSNSIVTERALREIYLEPFRLAVKYANPVCIMSSYNKVNGTHCSQSKKLLDDILREEWGWDGMITSDWFGTYSSADAIKNGLDIEFPGPTKWRKSELISHLISSKEGISEEDVNTRVRNVLKMIKFAVDNKDKTGIIENGPESDANNTPETAAKLRKIAADSIVLLKNENNVLPLSKDESIVVIGPNAKTKFMSGGGSASLNPYYVVPIYDGIKSKLGKDPEYSIGCTSNKTLNGLWEACVIDPSKGNQADNIGAQAIFYTKPVEDRSPDEKPIDSTTIKQSYLTLFDYKNPAVEESNPLFYVDFEGYYTPEEDGDYEIGLQVYGTALLFIDGELVVDNKTKQTKGTFCFSAGTIEEKAIVSMKAGKSYKFRVEYGSGPTSQTASDFGAGGMQVGIAKKIDENEEIAHAAQLAKEHDKVVLCIGLNGEWESEGYDRENMTLPKKTNDLVRAVLRANPNTVVVNQSGTPVEMPWISECNALLQCWYGGNELGNAVADIAFADVVPSAKLSLSWPFKNEDNPAYLNFATESGRVLYGEDVFVGYRFYEKLQRQVAFPFGYGLSYTTFTFENLEVTADESKEILSVQLDVTNSGSKYAGAEVVQVYVAPTKSGITRPVKELKGFKKVYLEPNETKKVSLELPLKDSISYFEEYHNKWCAEAGEYQLLAGSSSDDTQLISSFELSKTFYWKGL) had the same hydrolyzing ability and could hydrolyze sesame meal into sesaminol glycosides.,

[0087] Also, Smbg (SEQ ID NO: 5, MTFDIEKVLSELTTNEKISLIAAEDFWHTTPIKRLDIPSVRVSDGPNGIRGTKFFNSVPSAAFPNGTALASTFDKELLKEVGARMADEAIQKNAGVILGPTINIQRGPLGGRGFESFSEVPYLSGIAASCIVNGIQSRGVAATLKHFVCNDLEDQRMSSNSIVTCRALREIYLEPFKLAVKYSDPQCIMTSYNKVNGVHCSNSKNLLIDILRDEWKWGGMVMSDWFGTYSVDSIKNGLDIEFPGPSKWRSLDLLKSNLDSKAGITISNIDDCVRHVLKLVHYVSENSKKTQIKDHGPETTLNNTEHMSKHLRKVASESIVLLKNVDDILPLKKESSVVVIGPNAKAKSYSGGGSASLQPYYVITPYEGICEKIGRNVEYTAGCDSRKTLSGLIEAMVVDPCQPAEGDNIGIIAQYFMDPANQRSADVEPFDTHRVTQSYVTLFDYTHPNIDPIMPFFYIHFEGFFTPEEDGEYIFGVQVFGTALFYIDDKLEIDNKTHQTKGSFCFGAGTREETCEKYLVKGHQYRIRIEYGSGPTSSVAADFGYGGIQVGFAKKLNADEEIARAVQLAKTNDNVILCIGLNGEWESEGYDRENMSLPKNNDRLISAVLTANPNTIVVNQSGMPVELPWVDQCKALLQCWYGGNELGNAIADVLYGDVVPSGKLSISWPYMCHDNPAFLNFKTESGRVLYGEDIYVGYRFYEKVRRQVAFPFGHGLSYTTFRFDDLIVSIDETLDLLETSLNVTNTGDTIAGKEVIQVYVSHNESTIGRPIKELKGFQKVFLKPNETKKVTMKLSLKDSISYFDEEKQLWCATEGIYQILVGSSSKDIKLTERFDVNKTSYWKGL), having 61%, 58 and 57% sequence identity, Hobg (SEQ ID NO: 6,MPIDFNVDRLLTELTLDEKLSLLAGQDWWHTAAIERLNIPSVRVSDGPNGIRGTRFFACVPSACFPNGTALASTFNEEILESAGELMALEAKHKGAKVILGPTANIQRGPLGGRGFESFSEDPYLSGVATAAVVNGIQKSNEIAATVKHFVCNDMEHERFSSNSIVSERALREIYLEPFRLAVKHAQPKLFMTSYNKLNGIHCSSSKKLLQDILRNDWNSGATVISDWFGITDIVDSIQNGLDIEFPGPTRYRKPEILKNLLMCKTETREGGQFSIEHIDARARKVLELVKYFVEAEQSTDFPTNEDDHNNTFETAQFLRRLGNETIVLLKNETKLLPLDKKDDIVVIGPNAKAKNSSGGGCAALNGYYTISPLEGIANVTQRKTGDIPYTKGCDNHKNLSNLIEQCTNDADPEKKGAEMNFYTQPREVRGKEKPFDSYIIDQSFITLFDYKHEKVDEKKRLFYCTIEGYFIPKEDGDFEFQCQVLGTALFYIDDKLVINNKDDQTAGNFGFGSGTAPKNNIVTLEKGRKYKIFVDYGSGVTSKLSQSIAAGALQIGVNKVIDAEAEIKKAAELASKHDKVILVIGLNGEIESEGYDRDNMQLPRRTNDLVTAVLKANPNTVIVNQSGTPVEFPWLQQATTLLQAYYGGNELGNSIADVVFGDANPSGKLSLSWPLKNEDNPAYLNFKTVMGRVLYGEDIYVGYRFYEKLQRQVAYPFGHGLSYTTFKFNELDVSGDDESLKVELSVANTGKVDGKEVVQVYVARTSPSAVPRPVKELKKFKKVALKAGESAKVELTLSVKDSCSYFDEFHNQWHLEAGKYQVLVGSSSDDIHLIGDFEVKESEFWLGL) and Clavbg (SEQ ID NO:7,MADIDVEKVLSELTLAEKIGLTAGVDFWHTYKVERLGVPTLRLSDGPNGVRGTKFVNGAPSACFPCGTGLASTFNKDLLYKAGRLMADEAKHKSAHVILGPTTNMQRGPLGGRGFESFSEDPHLAGMASASIVKGMQDNDIAATIKHFVCNDLEHERNSSDAIVTERALREIYLEPFRLAVKYADPKSFMTAYNKVNGEHVSQSHRILEQILREEWNWDGLVMSDWYGAYTAKESLTNGLDLEMPGPSGMRTVQNISHMVNSRELNIKYLDERVRNVLKLVKWCARSKLEERGPETTENNTPETRALLNKIASELVVLLKNNDSVLPLKKEESIAVIGPNAKFAAYCGGGSASLLSYYTTTPYDAIKEKLGHEPKYAVGCYAHQMLPGFSLSPYTKNPVTGKSGVNCKLYNDAPGTKNRRQFDEFDITMSPIILFDYRHPAIVDELFYMDITGDLTPEESGEYEFSLTVSGTAQLFIDDKLVIDNKNSQTLGTAFFGTGTIEMKQKVPLDAGKTYNVRVEFGSSKTSKLRPLSVISFGGCVSIGMCKVIDPKEEIAKAVELAKSVDKVVLLIGLNAEWESEGFDRPDMELPLLTNDLVEAVLAANPNTVVVNQSGTPVEMPWLSKANALVHAWYGGSEAGNAIANVLFGDVNPSGKLSLSWPFKNSDNPAYLNFHTERGRVLYGEDIYIGYRFYDKLQRRVAFPFGYGLSYTTYKYSDLNVTVNEEDDSLTASVTVENTGSKDGAETVQFYVAPKTSEVARPVKELKGFDKVFVKAGEKATAKVQLSLKDSASFFDEYHDKWSLEKGTYEVQVGKSSDDVELIQEFKVKESKLWSGL) also shows some hydrolytic ability.,

[0088] Recovery of sesaminol from the enzyme reaction solution In this test, due to the poor water solubility of sesaminol, it was placed in a low-temperature environment to promote the precipitation of sesaminol. In this example, 60% methanol extract and hot water extract were used as reaction substrates, and the reaction was carried out under the same enzyme addition amount and reaction conditions. The precipitate obtained by the low-temperature precipitation method was used as the sesaminol concentrate to evaluate the feasibility of recovering sesaminol by the precipitation method. The results are shown in Table 4 below. The yield was calculated by (sesaminol concentrate / raw material weight) x 100%. The two yields were 11.1% and 10.7% respectively, and there was no significant difference. However, the sesaminol content differed by a factor of two, indicating that the purity of the reaction raw material affects the purity of the final product. In the low-temperature precipitation method, both recovery rates reached over 95%, indicating that there was basically no loss of sesaminol during the recovery process, showing that it is a feasible method.

[0089]

Table 5

[0090] Establishment of a method for preparing sesaminol from sesame meal extract Based on the above test, the feasibility of recovering sesaminol by the low-temperature precipitation method was confirmed. Next, the actual flow for preparing sesaminol was studied. As shown in Figures 8 and 9, the first method (Figure 8) used defatted sesame meal raw material, extracted it with 60% methanol to prepare sesame meal extract, then added an enzyme solution amount of 200 U / g extract, reacted at pH 6.5 and 40 °C for 16 hours, and then recovered sesaminol by the low-temperature precipitation method. The sesaminol content of the final product was 42.4% and the total recovery rate was 96.9%. The second method (Figure 9) added 10 times the amount of water to the sesame meal raw material, extracted it under the conditions of 121 °C and 60 minutes to prepare a water extract, then added an enzyme solution amount of 100 U / g water extract, reacted at pH 6.5 and 40 °C for 16 hours, and further recovered sesaminol by the low-temperature precipitation method. The sesaminol content of the final product was 21.2% and the total recovery rate was 85.0%. As can be seen from this, the first method is more ideal, while the second method is more feasible in practice.

[0091] In short, the method for producing sesaminol or sesaminol glycoside provided by the present invention not only shortens the manufacturing process time, but also increases the production amount of sesaminol and can reduce the production cost, thus meeting the requirements of industrial applicability.

[0092] All ranges provided herein are intended to include each specific range within a given range and combinations of sub-ranges between given ranges. Also, any range explicitly described herein includes its endpoints unless otherwise explicitly stated. Thus, the range of 1 to 5 includes, in particular, 1, 2, 3, 4, and 5, and sub-ranges such as 2 to 5, 3 to 5, 2 to 3, 2 to 4, 1 to 4, etc.

[0093] The present invention has been described in detail above. However, the above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. That is, equivalent changes and modifications made based on the claims of the present invention fall within the scope of patent protection of the present invention.

Claims

**Claim 1** A method for producing sesaminol or a sesaminol glycoside, comprising reacting a protein selected from the group consisting of the following (1) to (3) with a substrate sesaminol glycoside having at least one glycosidic bond to catalyze the hydrolysis of the at least one glycosidic bond. (1) A protein consisting of the amino acid sequence of SEQ ID NO: 1; (2) A protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having an activity of catalyzing the hydrolysis of the at least one glycosidic bond; (3) A protein consisting of an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 1 and having an activity of catalyzing the hydrolysis of the at least one glycosidic bond **Claim 2** The method according to claim 1, wherein the substrate sesaminol glycoside is selected from the group consisting of sesaminol 2′-O-β-D-glucopyranoside (sesaminol monoglucoside, SMG), sesaminol 2′-O-β-D-glucopyranosyl(1-2)-O-β-D-glucopyranoside (sesaminol (1-2) diglucoside, SDG(1,2)), sesaminol 2′-O-β-D-glucopyranosyl(1-6)-O-β-D-glucopyranoside (sesaminol (1-6) diglucoside, SDG(1,6)) and sesaminol 2′-O-β-D-glucopyranosyl(1-2)-O-(-β-D-glucopyranosyl(1-2))β-D-glucopyranoside (sesaminol triglucoside, STG). **Claim 3** The method according to claim 1, wherein the substrate sesaminol glycoside is a -60% (v / v) methanol crude extract. **Claim 4** The method according to claim 1, wherein the sesaminol or sesaminol glycoside is selected from the group consisting of sesaminol (1-6) diglucoside (SDG(1,6)), sesaminol (1-2) diglucoside (SDG(1,2)) and sesaminol. **Claim 5** The method according to claim 1, wherein the temperature at which the protein reacts with the substrate sesaminol glycoside is in the range of 37 to 45°C. **Claim 6** The method according to claim 1, wherein the reaction between the protein and the substrate sesaminol glycoside is carried out under the conditions of pH 5.5 to 6.

5. **Claim 7** The method according to claim 1, wherein the reaction time between the protein and the substrate sesaminol glycoside is 16 hours.

8. The method according to claim 1, wherein the at least one glycosidic bond is selected from the group consisting of a glycosidic bond between glucose bonded to the 2'-position of sesaminol and an aglycone, a β-1,6-glycosidic bond of gentiobiose bonded to the 2'-position of sesaminol, a β-1,2 bond of sophorose bonded to the 2'-position of sesaminol, a β-1,6-glycosidic bond and a β-1,2 bond of a branched trisaccharide bonded to the 2'-position of sesaminol.

9. A method for producing sesaminol or a sesaminol glycoside, comprising reacting an enzyme derived from a non-human transformed cell into which a polynucleotide selected from the group consisting of the following (1) to (5) has been introduced in a host cell with a substrate sesaminol glycoside having at least one glycosidic bond to catalyze hydrolysis of the at least one glycosidic bond. (1) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 1; (2) A polynucleotide encoding a protein consisting of an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 1 and having an activity of catalyzing hydrolysis of the at least one glycosidic bond; (3) A polynucleotide encoding a protein consisting of an amino acid sequence having at least 60% sequence identity to the amino acid sequence of SEQ ID NO: 1 and having an activity of catalyzing hydrolysis of the at least one glycosidic bond; (4) A polynucleotide that hybridizes under highly stringent conditions with a polynucleotide consisting of a base sequence complementary to the polynucleotide of the protein consisting of the amino acid sequence of SEQ ID NO: 1 and encodes a protein having an activity of catalyzing hydrolysis of the at least one glycosidic bond; (5) A polynucleotide consisting of the base sequence of SEQ ID NO: 2

10. The method according to claim 9, wherein the polynucleotide is inserted into an expression vector.

11. The method according to claim 9, wherein the non-human transformed cell is selected from the group consisting of transformed plant cells, transformed animal cells, transformed insect cells, transformed Escherichia coli, transformed Bacillus subtilis, transformed Actinomycetes, transformed bacteria, transformed yeast, and transformed filamentous fungi.

12. The method according to claim 9, wherein the substrate sesaminol glycoside is selected from the group consisting of sesaminol 2'-O-β-D-glucopyranoside (sesaminol monoglucoside, SMG), sesaminol 2'-O-β-D-glucopyranosyl(1-2)-O-β-D-glucopyranoside (sesaminol(1-2)diglucoside, SDG(1,2)), sesaminol 2'-O-β-D-glucopyranosyl(1-6)-O-β-D-glucopyranoside (sesaminol(1-6)diglucoside, SDG(1,6)), and sesaminol 2'-O-β-D-glucopyranosyl(1-2)-O-(-β-D-glucopyranosyl(1-2))β-D-glucopyranoside (sesaminol triglucoside, STG).

13. The method according to claim 9, wherein the sesaminol or sesaminol glycoside is selected from the group consisting of sesaminol(1-6)diglucoside (SDG(1,6)), sesaminol(1-2)diglucoside (SDG(1,2)), and sesaminol.

14. The method according to claim 9, wherein the at least one glycosidic bond is selected from the group consisting of a glycosidic bond between glucose bonded to the 2'-position of sesaminol and an aglycone, a β-1,6-glycosidic bond of gentiobiose bonded to the 2'-position of sesaminol, a β-1,2-bond of sophorose bonded to the 2'-position of sesaminol, a β-1,6-glycosidic bond and a β-1,2-bond of a branched trisaccharide bonded to the 2'-position of sesaminol.

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