JP6916801B2 - Production method of sesaminol or sesaminol glycoside - Google Patents

Description

The present invention relates to a method for producing sesaminol or a sesaminol glycoside.

Sesame (Sesamum indicum) is an annual plant of the genus Sesamum in the family Pedaliaceae. Sesame is considered to be the oldest cultivated oil plant with a history of about 6000 years and has been cultivated all over the world. Sesame has been a valuable food since ancient times and is known as a healthy food. In particular, sesame seeds, oils obtained from sesame seeds, and sesame seed extracts are used. About 50% of the components contained in sesame seeds are lipids, and the main components thereof are triglycerides mainly composed of oleic acid and linoleic acid. In addition, sesame lignan is contained as a characteristic component in sesame seeds.

Lignan is a secondary metabolite of plants, which is a _{compound in which two molecules of a phenylpropanoid compound having a C 6} C ₃ skeleton are polymerized (8,8'-bonded) mainly via these 8-8'positions. .. Lignans are thought to contribute to the biological defense mechanism in plants. The lignan contained in sesame is called sesame lignan, and includes sesamin, sesaminol, sesamolin, pinoresinol, and sesamolinol (Non-Patent Document 1). Sesamin, which is one of sesame lignans, has various physiological activities, and a method of separating and purifying sesame seeds or sesame seed pomace has already been put into practical use.

Some of the sesame lignans are known to be present in plants as glycosides. Among sesame lignans, sesaminol has high antioxidant activity. Sesaminol exists as a sesaminol glycoside in sesame seeds, and sesaminol glycosides include sesaminol 2'-O-β-D-glucopyranoside (sesaminol monoglucoside, SMG) and 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-glucopyranocil (1-2)- There are O- (-β-D-glucopyranosyl (1-2)) β-D-glucopyranoside (sesaminol triglucoside, STG) and the like.

Sesaminol is also produced from sesamolin by acid catalysis during the refining process of sesame oil. Sesaminol has a strong antioxidant activity, and it has been reported that it has a physiological activity favorable to health (Non-Patent Document 2). The bioactivity of sesaminol is exerted in aglycones from which the sugar moiety has been removed.

In addition, sesame pomace produced during the production of sesame oil contains sesaminol glycosides, but they are not effectively utilized. If sesaminol glycosides can be hydrolyzed to obtain sesaminol, which is an aglycone, it is possible to produce sesaminol from inexpensive sesame squeezed lees. As a method for obtaining sesaminol from a sesaminol glycoside, a method using a fungal Aspergillus genus microorganism (Patent Document 1) and a fermentation method using a mixed culture of Bacillus genus and Enterococcus genus (Patent Document 2) are available. It is being developed. However, these methods are inefficient because it takes a long time to completely decompose the sesaminol glycoside. In addition, as a method for obtaining sesaminol from a sesaminol glycoside, a method using β-glucosidase derived from the bacterial KB0549 strain belonging to the genus Paenibacillus is disclosed (Patent Document 3 and Non-Patent Document 3). This enzyme cleaves the β-1,2- and β-1,6-glucoside bonds and the sesaminol-glucose β-glucoside bonds of sesaminol glycosides. When STG is used as a substrate, β-1,2-glucoside bonds are hydrolyzed preferentially over β-1,6-glucoside bonds.

JP-A-2005-23125 JP 2006-61115 Japanese Patent Application Laid-Open No. 2008-167712

Biochemical Systematics and Ecology 13, 133-139 (1985) J. Agric. Food Chem., 64,4908-4913 (2016) PLOS ONE, 8, e60538 (2013)

Under the above circumstances, a new method for producing sesaminol and / or sesaminol glycosides has been sought.

As a result of diligent studies to solve the above problems, the present inventors hydrolyze sesaminol triglucoside to produce sesaminol and sesaminol glycosides, which is a glycoside hydrolase derived from aspergillus. They have found that they have activity and have completed the present invention.

That is, the present invention is as follows.
(1) At least one of the substrate sesaminol glycosides is reacted by reacting a protein selected from the group consisting of the following (a) to (c) with a substrate sesaminol glycoside having at least one glucoside bond. A method for producing a produced sesaminol or a produced sesaminol glycoside, which comprises a step of hydrolyzing a glucoside bond.
(A) A protein consisting of the amino acid sequence of SEQ ID NO: 2;
(B) A substrate sesaminol glycoside consisting of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2 and having at least one glucosidic bond. A protein that has the activity of hydrolyzing the glycosidic bond of
(C) Activity of hydrolyzing the glucoside bond of the substrate sesaminol glycoside having 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 and having at least one glucoside bond. (2) The substrate sesaminol glycosides are sesaminol 2'-O-β-D-glucopyranoside (sesaminol monoglucoside, SMG) and sesaminol 2'-O-β-D-glucopyranocil (1). -2) -O-β-D-glucopyranoside (sesaminol (1-2) diglucoside ,, SDG (1,2)), sesaminol 2'-O-β-D-glucopyranocil (1-6) -O- β-D-glucopyranoside (sesaminol (1-6) diglucoside, SDG (1,6)), and sesaminol 2'-O-β-D-glucopyranocil (1-2) -O- (-β-D-) The method according to (1) above, which is any one selected from the group consisting of glucopyranosyl (1-2)) β-D-glucopyranoside (sesaminol triglucoside, STG).
(3) The method according to (1) or (2) above, wherein the substrate sesaminol glycoside is sesaminol triglucoside (STG).
(4) The above (1), wherein the produced sesaminol or produced sesaminol glycoside is any one or more selected from the group consisting of SDG (1,6), SDG (1,2) and sesaminol. ) Or the method according to (2).
(5) The at least one glucosidic bond is a glucosidic bond between glucose and aglycon bound at the 2'position of sesaminol, a β-1,6-glucoside bond of gentiobiose bound to the 2'position of sesaminol, and sesaminol. Selected from the group consisting of β-1,2 bonds of sophorose bound to the 2'position, or β-1,6-glucoside bonds or β-1,2 bonds of branched trisaccharides bound to the 2'position of sesaminol. The method according to any one of (1) to (4) above, which is any one of the above.
(6) An enzyme agent derived from a non-human transformed cell into which a polynucleotide selected from the group consisting of the following (a) to (e) has been introduced into a host cell, a substrate sesame having at least one glucosidic bond. A method for producing a produced sesaminol or a produced sesaminol glycoside, which comprises a step of hydrolyzing at least one glucoside bond of the substrate sesaminol glycoside by contacting with a nool glycoside.
(A) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;
(B) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(C) A substrate sesaminol glycoside consisting of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2 and having at least one glucosidic bond. A polynucleotide encoding a protein that has the activity of hydrolyzing the glycosidic bond of
(D) Activity of hydrolyzing the glucosidic bond of a substrate sesaminol glycoside having an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 and having at least one glucoside bond. A polynucleotide encoding a protein having
(E) A substrate sesame that is a polynucleotide that hybridizes under highly stringent conditions with a polynucleotide having a base sequence complementary to the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 and has at least one glucosidic bond. The method according to (6) above, wherein the polynucleotide encoding a protein having an activity of hydrolyzing the glucosidic bond of a nool glycoside (7) is the polynucleotide inserted into an expression vector.
(8) The method according to (6) or (7) above, wherein the transformed cells are selected from the group consisting of transformed plants, transformed Escherichia coli, transformed bacteria, transformed yeast, and transformed filamentous fungi.
(9) The substrate sesaminol glycoside is 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-glucopyranocil (1-2) -O- (-β-D-glucopyranocil (1--)) 2)) The method according to any one of (6) to (8) above, which is any one selected from the group consisting of β-D-glucopyranoside (sesaminol triglucoside, STG).
(10) The produced sesaminol or produced sesaminol glycoside is at least one selected from the group consisting of SDG (1,6), SDG (1,2) and sesaminol. )-(9).
(11) The at least one glucosidic bond is a glucosidic bond between glucose and aglycon bound at the 2'position of sesaminol, a β-1,6-glucoside bond of gentiobiose bound to the 2'position of sesaminol, and sesaminol. Selected from the group consisting of β-1,2 bonds of sophorose bound to the 2'position, or β-1,6-glucoside bonds or β-1,2 bonds of branched trisaccharides bound to the 2'position of sesaminol. The method according to any one of (6) to (10) above, which is any one of the above.
(12) A method for producing a produced sesaminol or a produced sesaminol glycoside, which comprises culturing a non-human transformant into which a polynucleotide selected from the group consisting of the following (a) to (e) has been introduced. ..
(A) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;
(B) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(C) A substrate sesaminol glycoside consisting of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2 and having at least one glucosidic bond. A polynucleotide encoding a protein that has the activity of hydrolyzing the glycosidic bond of
(D) Activity of hydrolyzing the glucosidic bond of a substrate sesaminol glycoside having an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 and having at least one glucoside bond. A polynucleotide encoding a protein having
(E) A substrate sesame that is a polynucleotide that hybridizes under highly stringent conditions with a polynucleotide having a base sequence complementary to the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 and has at least one glucosidic bond. The method according to (10) above, wherein the polynucleotide encoding a protein having an activity of hydrolyzing the glucosidic bond of a nool glycoside (13) is the polynucleotide inserted into an expression vector.
(14) The method according to (12) or (13) above, wherein the transformant is selected from the group consisting of transformed plants, transformed Escherichia coli, transformed bacteria, transformed yeast, and transformed filamentous fungi.
(15) The at least one glucosidic bond is a glucosidic bond between glucose and aglycon bound at the 2'position of sesaminol, a β-1,6-glucoside bond of gentiobiose bound to the 2'position of sesaminol, and sesaminol. Selected from the group consisting of β-1,2 bonds of sophorose bound to the 2'position, or β-1,6-glucoside bonds or β-1,2 bonds of branched trisaccharides bound to the 2'position of sesaminol. The method according to any one of (12) to (14) above, which is any one of the above.

The method of the present invention provides a new method for producing sesaminol and sesaminol glycosides.
In addition, various sesaminol glycosides can be produced by selecting the reaction conditions. In one embodiment of the present invention, the amount of sesaminol and / or sesaminol glycoside produced can be increased by selecting reaction conditions such as enzyme concentration. Further, according to one embodiment of the present invention, the amount of SDG (1, 2) produced can be selectively increased.

It is a figure of the cDNA sequence and the amino acid sequence of AOBGL11. It is a figure of the cDNA sequence and the amino acid sequence of AOBGL11. A: Optimal temperature of AOBGL11p using pNP-β-Glc as a substrate. B: Optimal pH of AOBGL11p using pNP-β-Glc as a substrate. C: Thermal stability of AOBGL11p using pNP-β-Glc as a substrate. D: It is a figure of the pH stability of AOBGL11p using pNP-β-Glc as a substrate. It is a figure of comparison of the genomic DNA sequence and the cDNA sequence of AOBGL11. It is a figure of comparison of the genomic DNA sequence and the cDNA sequence of AOBGL11. It is a figure of the hydrolysis reaction of sesaminol triglucoside. The reaction time was 1 hour. A: BGL11-1 crude enzyme solution diluted 10-fold, B: BGL11-1 crude enzyme solution without dilution, C: C-1 crude enzyme solution.

Hereinafter, the present invention will be described in detail. The following embodiments are examples for explaining the present invention, and the present invention is not intended to be limited to this embodiment. The present invention can be implemented in various forms as long as it does not deviate from the gist thereof. All documents cited in this specification, as well as published publications, patent publications and other patent documents shall be incorporated herein by reference. In addition, this specification includes the contents described in the specification and drawings of the Japanese patent application (Japanese Patent Application No. 2016-186066) which is the basis of the priority claim of the present application filed on September 23, 2016.

In the following, the sesaminol glycoside as a substrate may be referred to as a "substrate sesaminol glycoside", and the sesaminol glycoside as a product may be referred to as a "produced sesaminol glycoside". In addition, both are sometimes collectively referred to as "sesaminol glycosides".

"AOBGL11p" is a β-glucosidase of the glycoside hydrolase family 3 (GH3), the cDNA sequence of which is shown in SEQ ID NO: 1, the amino acid sequence of which is shown in SEQ ID NO: 2, and the genomic DNA sequence of which is shown in SEQ ID NO: 3.

1. 1. Method for Producing Sesaminol and / or Sesaminol Glycoside The present invention comprises a protein selected from the group consisting of the following (a) to (c) (hereinafter referred to as "protein of the present invention") and a sesaminol glycoside. Provided is a method for producing a sesaminol and / or sesaminol glycoside, which comprises a step of reacting a sugar body to hydrolyze at least one glucoside bond.
(A) A protein consisting of the amino acid sequence of SEQ ID NO: 2;
(B) In the amino acid sequence of SEQ ID NO: 2, 1 to 83 amino acids consist of an amino acid sequence deleted, substituted, inserted, and / or added, and hydrolyze at least one glucosidic bond of a sesaminol glycoside. Proteins with degrading activity;
(C) A protein having an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 and having an activity of hydrolyzing at least one glucosidic bond of a sesaminol glycoside.

The protein according to (b) or (c) above is typically a variant of the protein consisting of the amino acid sequence of SEQ ID NO: 2, for example, "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 4, Cold Spring Harbor Laboratory Press 2012 ”,“ Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997 ”,“ Nuc. Acids. Res., 10, 6487 (1982) ”,“ Proc. Natl. Acad . Sci. USA, 79, 6409 (1982) ”,“ Gene, 34, 315 (1985) ”,“ Nuc. Acids. Res., 13, 4431 (1985) ”,“ Proc. Natl. Acad. Sci. USA , 82, 488 (1985) ”etc., including those that can be artificially obtained by using the site-specific mutagenesis method.

"In the amino acid sequence of SEQ ID NO: 2, 1 to 83 amino acids consist of the deleted, substituted, inserted, and / or added amino acid sequence and hydrolyze at least one glucoside bond of the sesaminol glycoside. Examples of the "active protein" include, in the amino acid sequence of SEQ ID NO: 2, 1 to 83, 1 to 80, 1 to 75, 1 to 70, 1 to 65, 1 to 60, 1 to 1. 55, 1-50, 1-49, 1-48, 1-47, 1-46, 1-45, 1-44, 1-43, 1-42, 1- 41, 1-40, 1-39, 1-38, 1-37, 1-36, 1-35, 1-34, 1-33, 1-32, 1- 31, 1 to 30, 1 to 29, 1 to 28, 1 to 27, 1 to 26, 1 to 25, 1 to 24, 1 to 23, 1 to 22 and 1 to 1 21, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1- 11 pieces, 1-10 pieces, 1-9 pieces (1 to several pieces), 1-8 pieces, 1-7 pieces, 1-6 pieces, 1-5 pieces, 1-4 pieces, 1-3 pieces, 1 A protein consisting of an amino acid sequence in which ~ 2 or 1 amino acid residue is deleted, substituted, inserted and / or added and has an activity of hydrolyzing at least one glucoside bond of a sesaminol glycoside. Can be mentioned. Generally, the smaller the number of deletions, substitutions, insertions and / or additions of the amino acid residues is, the more preferable.

Further, such proteins include the amino acid sequence of SEQ ID NO: 2, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98%. Above, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99. Examples thereof include proteins having an amino acid sequence having 8% or more or 99.9% or more sequence identity and having an activity of hydrolyzing at least one glucosidic bond of a sesaminol glycoside. Generally, the larger the value of the sequence identity is, the more preferable it is.

Here, "at least one glucosideic bond of a sesaminol glycoside" means that in a sesaminol glycoside, which is a glycoside in which a sugar is bound to sesaminol, between sesaminol, which is an aglycone, and a side chain. It means a glucoside bond and a glucoside bond in a side chain. Further, "the activity of hydrolyzing the glucoside bond of a substrate sesaminol glycoside having at least one glucoside bond" is an aglycon in the sesaminol glycoside which is a glycoside in which a sugar is bound to sesaminol. It means the activity of cleaving (hydrolyzing) at least one of the glucosidic bond between the sesaminol and the side chain and the glucosidic bond in the side chain. Examples of glucosidic bonds in the side chain are β-1,6-glucosidic bond of gentiobiose bound to the 2'position of sesaminol, β-1,2-glucosidic bond of sophorose bound to the 2'position of sesaminol, and sesame. Examples thereof include the β-1,2-glucosidic bond of the branched trisaccharide bound to the nol 2'position and / or the β-1,6-glucosidic bond of the branched trisaccharide bound to the sesaminol 2'position. In certain embodiments, all glucosidic bonds are hydrolyzed to yield sesaminol from sesaminol glycosides. In another embodiment, the β-1,6-glucoside bond of the branched trisaccharide bound to the sesaminol 2'position is preferentially hydrolyzed. In this way, a produced sesaminol glycoside in which only some sugars are cleaved or sesaminol in which all sugars are cleaved is produced.

The activity of hydrolyzing the glucosidic bond of the substrate sesaminol glycoside having at least one glucosidic bond comprises the protein of the present invention and, for example, STG, SDG (1,2), SDG (1,6), SMG. At least one substrate sesaminol glycoside selected from the above is reacted to purify the obtained reaction product (sesaminol and / or sesaminol glycoside), and the purified product is subjected to liquid chromatography (Liquid Chromatografy). : It can be confirmed by analysis by a known method such as LC).

In one embodiment, the present invention provides a method for producing sesaminol, which comprises the step of cleaving all glucosidic bonds of a sesaminol glycoside. In another embodiment, the invention presents a specific bond of a sesaminol glycoside, eg, a glucosidic bond between glucose bound to the sesaminol 2'position and an aglycon, a β of gentiobiose bound to the sesaminol 2'position. -1,6-glucosidic bond, β-1,6-glucoside bond of sophorose bound to the 2'position of sesaminol and / or β-1,6-glucosidic bond of the branched trisaccharide bound to the 2'position of sesaminol or Provided is a method for producing a sesaminol and / or a sesaminol glycoside, which comprises a step of cleaving a β-1,2 bond. In the present invention, when the branched trisaccharide bound to the sesaminol 2'position is hydrolyzed, the β-1,6-bond is preferentially hydrolyzed over the β-1,2-bond.

"In the amino acid sequence of SEQ ID NO: 2, 1 to 83 amino acid residues have been deleted, substituted, inserted and / or added" means any position in the same sequence and any position in the 1 to 83 amino acid sequence. Means that there are deletions, substitutions, insertions and / or additions of 1 to 83 amino acid residues, and two or more of the deletions, substitutions, insertions and additions may occur simultaneously.

Examples of mutually replaceable amino acid residues are shown below. Amino acid residues contained in the same group can be replaced with each other.

Group A: leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine;
Group B: aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosveric acid;
Group C: asparagine, glutamine;
Group D: lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid;
Group E: Proline, 3-Hydroxyproline, 4-Hydroxyproline;
Group F: serine, threonine, homoserine;
Group G: Phenylalanine, tyrosine.

The protein of the present invention can be obtained by expressing a polynucleotide encoding the same (see "Polynucleotide of the present invention" described later) in an appropriate host cell, or the like, using the Fmoc method (fluorenylmethyl). It can also be produced by a chemical synthesis method such as the oxycarbonyl method) or the tBoc method (t-butyloxycarbonyl method). Also, manufactured by AAPPTec LLC, Perkin Elmer Inc. Manufactured by Protein Technologies Inc. Chemical synthesis can also be performed using a peptide synthesizer manufactured by Peptide Synthes, PerSeptive Biosystems, Applied Biosystems, SHIMADZU CORPORATION, or the like.

In the present invention, the "sesaminol glycoside" is a glycoside in which a sugar is bound to sesaminol.

Examples of sesaminol and sesaminol glycosides are represented by the following general formula (I). The 2'position of sesaminol is shown in the formula. In the present invention, "branched trisaccharide" refers to what constitutes a trisaccharide among those represented by the following general formula (I) is added to a portion of the R _1.

Examples of sesaminol glycosides include, but are not limited to, SMG, SDG (1,2), SDG (1,6) and STG.

In 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: 2 and / or a protein consisting of a variant thereof. In another embodiment, a sesaminol glycoside is available as a substrate. Here, examples of sesaminol glycosides are as described above. In one embodiment of the present invention, the reaction conditions are adjusted by adjusting the amount of the enzyme added to the reaction, adding an organic solvent to the reaction solution, or adjusting the reaction temperature by using the enzyme of the present invention. By adjusting, rare sesaminol glycosides such as SDG (1,2) and SDG (1,6) are produced using STG, which is the most abundant sesaminol glycoside in sesame oil pomace, as a substrate. Is also possible.

In the present invention, the amount of enzyme, substrate / enzyme ratio, temperature, pH, presence / absence of solvent, reaction time, etc. of the reaction using the sesaminol glycoside as a substrate can be appropriately adjusted by those skilled in the art, and by adjusting these, sesame It is possible to control the degree of decomposition to what extent the sugar bound to the solvent glycoside is cleaved.

According to the method for producing a sesaminol and / or a sesaminol glycoside according to the present invention, at least one glucoside bond of the sesaminol glycoside is hydrolyzed.

When the enzyme of the present invention is used, sesaminol glycosides and / or sesaminol are produced.
In addition, the enzyme of the present invention can preferentially cleave a specific bond in the sesaminol glycoside molecule, thereby obtaining, for example, SDG (1, 2). In the present application, "preferentially cleaving a specific bond in a sesaminol glycoside molecule" means selectively and preferentially hydrolyzing a specific glucoside bond in a sesaminol glycoside. For example, when STG is used as a substrate, SDG (1, 2) is produced in which the β-1,6 bond of the branched trisaccharide bound to the sesaminol 2'position of STG is preferentially hydrolyzed. Examples of the "specific bond in the sesaminol glycoside molecule" include β-1,6 bonds of the branched trisaccharide bound to the sesaminol 2'position.

In the method for producing sesaminol and / or sesaminol glycoside of the present invention, the sesaminol glycoside as a starting material is an appropriate solvent (aqueous solvent such as water or alcohol, ether and sesame seeds or sesame oil squeezed lees). Extraction with organic solvents such as acetone), ethyl acetate and other organic solvents: water gradient, high performance liquid chromatography (HPLC), ultra-high performance liquid chromatography (Ultra (High) Performance Liquid chromatografy: UPLy) It can be obtained by extracting and purifying by a known method such as). Alternatively, the sesaminol glycoside as a starting material may be a commercially available product. Examples of the sesaminol glycoside used as the starting material of the present invention include STG, SDG (1,2), SDG (1,6), SMG and the like, and β-1,2-glucoside bond or β-1, 6 Glucoside bonds and / or cleavage of glucoside bonds with sesaminol, which is an aglycone, results in SDG (1,2), SDG (1,6), SMG, sesaminol and the like.

In the method for producing a sesaminol and / or a sesaminol glycoside according to the present invention, the protein of the present invention is reacted with the sesaminol glycoside to hydrolyze at least one glucoside bond of the sesaminol glycoside. Includes steps. The method of the present invention may further include a step of purifying the sesaminol and / or sesaminol glycoside of the present invention produced in the above step. The sesaminol and / or sesaminol glycoside of the present invention can be extracted with a suitable solvent (aqueous solvent such as water or organic solvent such as alcohol, ether, acetone), ethyl acetate and other organic solvents: gradient of water. , High Performance Liquid Chromatography (HPLC), Ultra High Performance Liquid Chromatography (ULTRA (High) Performance Liquid Chromatography: UPLC) and the like.

The method for producing a sesaminol glycoside and / or sesaminol according to the present invention can be carried out under the condition that an organic solvent is added to a reaction solution containing a substrate. The organic solvent can be in the range of 1% to 20%, preferably 5% to 15%, 6 to 12%, and more preferably 8% with respect to the total amount of the reaction solution. The organic solvent may be generally available, preferably mixed with water at an arbitrary ratio, and acetonitrile or the like can be used. The organic solvent may be added to the reaction solution in advance, or may be added in the middle of the reaction.

As used herein, the term "polynucleotide" means DNA or RNA.

Examples of the polynucleotide encoding the protein consisting of the amino acid sequence of SEQ ID NO: 2 include the polynucleotide consisting of the base sequence of SEQ ID NO: 1.

"In the amino acid sequence of SEQ ID NO: 2, 1 to 83 amino acids consist of an amino acid sequence deleted, substituted, inserted, and / or added, and hydrolyzes at least one glucosidic bond of the sesaminol glycoside. Examples of the "active protein" include those mentioned above.

As "a protein having an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 and having an activity of hydrolyzing at least one glucoside bond of a sesaminol glycoside", The above can be mentioned.

In the present specification, the "polynucleotide that hybridizes under highly stringent conditions" refers to, for example, a polynucleotide having a base sequence complementary to the base sequence of SEQ ID NO: 1 or an amino acid sequence of SEQ ID NO: 2. A polynucleotide obtained by using a colony hybridization method, a plaque hybridization method, a southern hybridization method, or the like, using all or a part of a polynucleotide having a base sequence complementary to the base sequence to be used as a probe. Hybridization methods include, for example, "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 4, Cold Spring Harbor, Laboratory Press 2012", "Ausubel, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997". You can use the method described in.
As used herein, the term "high stringent condition" refers to, for example, 5xSSC, 5xDenhardt solution, 0.5% SDS, 50% formamide, 50 ° C., or 0.2xSSC, 0.1% SDS. The conditions are 60 ° C., or 0.2xSSC, 0.1% SDS, 62 ° C., or 0.2xSSC, 0.1% SDS, 65 ° C., but the conditions are not limited thereto. Under these conditions, it can be expected that DNA having higher sequence identity can be efficiently obtained as the temperature is raised. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect the stringency of hybridization, and those skilled in the art can appropriately select these factors. By doing so, it is possible to achieve similar stringency.

When a commercially available kit is used for hybridization, for example, Alkaline Direct Labeling and Detection System (GE Healthcare) can be used. In this case, the membrane was first washed with 0.1% (w / v) SDS under conditions of 55-60 ° C. after incubation with the labeled probe overnight according to the protocol attached to the kit. After washing with a buffer, hybridized DNA can be detected. Alternatively, when preparing a probe based on all or part of the base sequence complementary to the base sequence of SEQ ID NO: 1 or the base sequence complementary to the base sequence encoding the amino acid sequence of SEQ ID NO: 2, it is commercially available. When the probe is labeled with digoxygenin (DIG) using a reagent (for example, PCR labeling mix (Roche Diagnostics)), hybridization is performed using a DIG nucleic acid detection kit (Roche Diagnostics). Can be detected.

As a polynucleotide capable of hybridizing other than the above, the DNA of the base sequence of SEQ ID NO: 1 or the amino acid sequence of SEQ ID NO: 2 is encoded when calculated using the default parameters by the homology search software of BLAST. DNA and 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99. DNA having 7% or more, 99.8% or more, or 99.9% or more sequence identity can be mentioned.

The sequence identity of amino acid sequences and base sequences is determined by the algorithm BLAST (Basic Local Element Search Tool) by Carlin and Arthur (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc Natl Acad Sci USA 90: It can be determined using 5873, 1993). When using BLAST, use the default parameters of each program.

The above-mentioned polynucleotide of the present invention can be obtained by a known genetic engineering method or a known synthetic method.

The polynucleotide of the present invention may further contain a polynucleotide consisting of a base sequence encoding a secretory signal peptide. Preferably, the polynucleotide consisting of the nucleotide sequence encoding the secretory signal peptide is contained at the 5'end of the polynucleotide of the present invention. The polynucleotide from the secretory signal peptide and the nucleotide sequence encoding it is the same as described above.

The polynucleotides of the invention are preferably introduced into the host in the state of being inserted into a suitable expression vector.

Suitable expression vectors are usually
(I) A promoter that can be transcribed in the host cell;
(Ii) The polynucleotide of the invention bound to the promoter; and (iii) for transcription termination and polyadenylation of the RNA molecule, are configured to include an expression cassette containing a signal that functions in the host cell as a component. ..

Examples of the method for producing an expression vector include, but are not limited to, a method using a plasmid, phage, cosmid, or the like.

The specific type of the vector is not particularly limited, and a vector that can be expressed in the host cell can be appropriately selected. That is, if a promoter sequence is appropriately selected in order to reliably express the polynucleotide of the present invention according to the type of host cell, and a vector in which this and the polynucleotide of the present invention are incorporated into various plasmids or the like is used as an expression vector. good.

The expression vector of the present invention contains an expression control region (eg, promoter, terminator and / or origin of replication, etc.), depending on the type of host to be introduced. Conventional promoters (for example, trc promoter, tac promoter, lac promoter, etc.) are used as the promoter of the expression vector for bacteria, and for example, the glyceraldehyde triphosphate dehydrogenase promoter, PH05 promoter, etc. are used as the promoter for yeast. Examples of the promoter for filamentous fungi include amylase, trpC and the like. As an example of a promoter for expressing the target gene in plant cells, the enhancer sequences of the 35S RNA promoter of the Califlower mosaic virus, the rd29A gene promoter, the rbcS promoter, and the 35S RNA promoter of the Califlower mosaic virus are derived from agrobacterium. The mac-1 promoter added to the 5'side of the mannopine synthase promoter sequence of the above can be mentioned. Examples of promoters for animal cell hosts include viral promoters (eg, SV40 early promoter, SV40 late promoter, etc.). Examples of promoters that are inducibly activated by external stimuli include mouse mammary tumor promoters (MMTV) promoters, tetracycline-responsive promoters, metallothionein promoters, heat shock protein promoters, and the like.

The expression vector preferably contains at least one selectable marker. Such markers include auxotrophic markers (ura5, niaD), drug resistance markers (hygromycin, zeocin), genetisin resistance gene (G418r), copper resistance gene (CUP1) (Marin et al., Proc. Natl. Acad. Sci. USA, vol. 81, p. 337, 1984), Cerlenin resistance gene (fas2m, PDR4) (Atsushi Inokoshi et al., Biochemistry, vol. 64, p. 660, 1992; Hussain et al. , Gene, vol. 101, p. 149, 1991) etc. are available.

The method for producing the transformant of the present invention (production method) is not particularly limited, and examples thereof include a method of introducing an expression vector containing the polynucleotide of the present invention into a host for transformation. As the cells or organisms to be transformed, various conventionally known cells or organisms can be preferably used. Examples of cells to be transformed include bacteria such as Escherichia coli, yeast (budding yeast Saccharomyces cerevisiae, fission yeast Schizosaccharomyces pombe), filamentous fungi (Aspergillus oryzae, Aspergillus sojae), plant cells, and humans. Excluded animal cells and the like can be mentioned. Suitable culture media and conditions for the above host cells are well known in the art. The organism to be transformed is also not particularly limited, and examples thereof include various microorganisms or plants or animals other than humans exemplified in the above host cells. The transformant is preferably a filamentous fungus, yeast or plant. As the host used for transformation, one that produces any of the sesaminol glycosides can also be used. Genes required for the production of at least one sesaminol glycoside not only in plants that originally produce at least one sesaminol glycoside, such as sesame, but also in cells or organisms that do not originally produce sesaminol glycosides. Can be used as a host. Examples of the "gene required for the production of sesaminol glycosides" include genes having sesaminol glycoside synthesis activity described in JP-A-2006-129728.

As a method for transforming a host cell, a commonly used known method can be used. For example, the electroporation method (Mackenxie, DA et al., Appl. Environ. Microbiol., Vol. 66, p. 4655-4661, 2000), the particle delivery method (Japanese Patent Laid-Open No. 2005-287403 "Breeding method for lipid-producing bacteria" ”), Spheroplast method (Proc. Natl. Acad. Sci. USA, vol.75, p. 1929, 1978), Lithium acetate method (J. Bacteriology, vol. 153, p. 163, 1983). ), Methods in yeast genetics, 2000 Edition: A Cold Spring Harbor Laboratory Course Manual, etc.), but not limited to these. In addition, when introducing a gene into a plant or a tissue or cell derived from a plant, for example, the Agrobacterium method (Plant Molecular Biology Mannual, Gelvin, SBet al., Academic Press Publishers), the particle gun method, or the PEG method. , Electroporation method and the like can be appropriately selected and used.

Method for producing sesaminol glycoside and / or sesaminol of the present invention using an enzyme agent derived from non-human transformed cells The protein of the present invention is expressed in a host cell and the cells are disrupted to cause the present invention. Protein can be obtained. By reacting the protein of the present invention with the substrate sesaminol glycoside, the sesaminol glycoside and / or sesaminol of the present invention can also be produced.
Specifically, sesaminol can be produced by contacting an enzyme agent derived from the transformed cell of the present invention with a sesaminol glycoside having at least one glucoside bond. It has been confirmed in Examples that the protein of the present invention exhibits the same activity when expressed in yeast as when expressed in Jiuqu.
The "enzyme agent derived from transformed cells" is not limited as long as it is prepared using transformed cells and contains the protein of the present invention, but for example, the transformed cells themselves, the pulverized product of the transformed cells themselves, etc. The culture supernatant of the transformed cells itself, and purified products thereof. Therefore, the present invention uses an enzyme agent derived from a non-human transformed cell into which a polynucleotide selected from the group consisting of the following (a) to (e) has been introduced into a host cell, and at least one glucosidic bond is added. Provided is a method for producing a sesaminol and / or sesaminol glycoside, which comprises a step of hydrolyzing at least one glucoside bond by contacting with a sesaminol glycoside having. (A) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;
(B) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(C) In the amino acid sequence of SEQ ID NO: 2, 1 to 83 amino acids consist of an amino acid sequence deleted, substituted, inserted, and / or added, and hydrolyze at least one glucosidic bond of a sesaminol glycoside. Polynucleotides encoding proteins with degrading activity;
(D) A protein protein having an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 and having an activity of hydrolyzing at least one glucosidic bond of a sesaminol glycoside. Polynucleotide encoding;
(E) A polynucleotide that hybridizes under highly stringent conditions with a polynucleotide having a nucleotide sequence complementary to the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, and at least one of sesaminol glycosides. A polynucleotide encoding a protein that has the activity of hydrolyzing glucoside bonds

The polynucleotide selected from the group consisting of the above (a) to (e) is the polynucleotide of the present invention, and is the same as described above.

"Contact" means that an enzyme agent derived from the transformed cell of the present invention and a sesaminol glycoside having at least one glucosidic bond are present in the same reaction system or culture system, for example. Adding a sesaminol glycoside having at least one glucoside bond to a container containing an enzyme agent derived from the transformed cell of the present invention, and at least one glucosidic bond with the enzyme agent derived from the transformed cell of the present invention. It includes mixing with a sesaminol glycoside having, and adding an enzyme agent derived from the transformed cell of the present invention to a container containing a sesaminol glycoside having at least one glucosidic bond.

When the transformant is yeast or aspergillus, the yeast or aspergillus transformed with the polynucleotide of the present invention expresses a large amount of the protein of the present invention as compared with the wild type. Therefore, the expressed protein of the present invention reacts with sesaminol glycosides produced in yeast or aspergillus, and sesaminol and / or sesaminol glycosides are preferably contained in yeast or aspergillus cells or in culture medium. Is produced in the culture medium.

When the transformant is a plant, the plant to be transformed in the present invention is the whole plant, plant organs (for example, leaves, petals, stems, roots, seeds, etc.), plant tissues (for example, epidermis, phloem). , Soft tissue, wood, phloem bundle, palisade tissue, spongy tissue, etc.) or plant cultured cells, or various forms of plant cells (eg, suspended cultured cells), protoplasts, leaf sections, callus, etc. Also means. The plant used for transformation may be either a monocotyledonous plant or a dicotyledonous plant. Whether or not the polynucleotide of the present invention has been introduced into a plant can be confirmed by a PCR method, a Southern hybridization method, a Northern hybridization method, or the like. 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 or asexual reproduction of the plant. In addition, for example, seeds, fruits, cut ears, tubers, tubers, strains, callus, protoplasts, etc. can be obtained from the plant or its descendants, or clones thereof, and the plant can be mass-produced based on them. can. A plant transformed with the polynucleotide of the present invention (hereinafter, "plant of the present invention") contains a large amount of the protein of the present invention as compared with its wild type. Therefore, the protein of the present invention reacts with the sesaminol glycoside produced in the plant of the present invention, and sesaminol is produced in the plant. In addition, when the environment in the plant is not optimal for the hydrolysis reaction, the hydrolysis reaction of the glucosidic bond of the sesaminol glycoside is suppressed, and the sesaminol glycoside in which the glucosidic bond is maintained without being cleaved or A sesaminol glycoside is produced in which only a part of the glucosidic bond is cleaved.

The transformant of some aspects of the present invention or a culture medium thereof has a high content of sesaminol and / or sesaminol glycoside of the present invention as compared with the wild type thereof, and the extract or culture medium thereof has a high content. , Sesaminol and / or sesaminol glycosides of the present invention are contained in high concentrations. The extract of the transformant of the present invention can be obtained by disrupting the transformant using glass beads, a homogenizer, a sonicator, or the like, centrifuging the disrupted product, and collecting the supernatant thereof. can. When the sesaminol and / or sesaminol glycoside of the present invention is accumulated in the culture medium, after the completion of the culture, the transformant and the culture supernatant are prepared by a usual method (for example, centrifugation, filtration, etc.). By separating the cells, a culture supernatant containing the sesaminol and / or sesaminol glycoside of the present invention can be obtained.

The extract or culture supernatant thus obtained may be further subjected to a purification step. Purification of the sesaminol and / or sesaminol glycosides of the present invention can be carried out according to conventional separation and purification methods. The specific method is the same as described above.

The "sesaminol glycoside", the "sesaminol glycoside having at least one glucoside bond", and the "activity to hydrolyze at least one glucoside bond" are the same as described above.

For other general molecular biology methods, see "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol.4, Cold Spring Harbor Laboratory Press 2012", "Methods in Yeast Genetics, A laboratory manual (Cold Spring Harbor)". Laboratory Press, Cold Spring Harbor, NY) ”etc. can be referred to.

The sesaminol and / or sesaminol glycoside of the present invention thus obtained can be used for producing, for example, foods, sweeteners, flavors, pharmaceuticals, industrial raw materials (raw materials such as cosmetics and soaps) according to a conventional method. It can be used for such purposes.

Examples of foods include foods for specified health use, foods with nutritional function, foods with functional claims, nutritional supplements, health foods, functional foods, foods for infants, foods for the elderly and the like. In the present specification, food is a general term for solids, fluids, and liquids, and mixtures thereof, which are edible.

All documents cited in this specification, as well as published publications, patent publications and other patent documents shall be incorporated herein by reference.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

[Example 1]
Search for β-glucosidase gene of aspergillus AO090701000244 (CDS sequence: SEQ ID NO: 1, estimated amino acid sequence: SEQ ID NO: 2, The ORF sequence: SEQ ID NO: 3 and the genomic DNA sequence: SEQ ID NO: 4) were found. I decided to clone this as AOBGL11. The cDNA sequence and amino acid sequence of AOBGL11 are shown in FIG.

Cloning of genomic DNA of AOBGL11 The following primers were designed for cloning AOBGL11.

AOBGL11-F:
5'-ATGCCTCGTCTAGACGTCGAGAA-3' (SEQ ID NO: 4)
AOBGL11-R:
5'-TCACAGACCCAACCAGTAGCGA-3' (SEQ ID NO: 5)

Aspergillus Aspergillus oryzae var.Brunneus (IFO30102) liquid medium (per 1L conidia of glucose 20g, bacto tryptone 1g, yeast extract _{5g, NaNO 3 1g, K 2} HPO 4 0.5g, MgSO 4 · 7H 2 O 0 _.5G, was inoculated into _{FeSO 4 · 7H 2 O 0.01g)} 10ml, and cultured for 1 day at 30 ° C.. Bacterial cells were collected by filtration, ground in liquid nitrogen, and then genomic DNA was prepared using DNeasy Plant Mini Kit (QIAGEN).
PCR was performed with KOD-plus (Toyobo) using the primers AOBGL11-F and AOBGL11-R using genomic DNA as a template. The obtained DNA fragment of about 2.57 kbp was cloned using Zero Blunt TOPO PCR Cloning Kit (Invitrogen) to obtain 11 g of plasmid pCR-AOBGL.

[Example 2]
Production of AOBGL11p by aspergillus Construction of vector for expressing aspergillus aspergillus DNA fragment obtained by digesting the aspergillus vector pUNA (Liquor Research Institute) with the restriction enzyme SmaI and the plasmid pCR-AOBGL11g were digested with the restriction enzyme EcoRI and the end was Blunting. A DNA fragment of about 2.57 kbp obtained by smoothing with Kit (Takarabio) was ligated to obtain 11 g of plasmid pUNA-AOBGL.

Transformation of Jiuqu The transformation of Jiuqu was carried out as follows.
Aspergillus oryzae niaD300 strain (Liquor Research Institute) was used as a host. The host strain was inoculated on a PDA plate and cultured at 30 ° C. for about 1 week. To obtain a conidia suspension, 0.1% tween80, 0.8% NaCl was added and the conidia were suspended. After filtering with Miracross, the splinters were collected by centrifugation, washed with 0.1% Tween80, 0.8% NaCl, and suspended in sterile water. ・ BR> B spawns were placed on a CD plate (per 1 L). _{NaNO 3 6g, KCl 0.52g, KH} 2 PO 4 1.52g, glucose _{10g, 1M MgSO 4 2ml, Trace} element solution ( per _{1L, FeSO 4 · 7H 2 O} 1g, ZnSO 4 · 7H 2 O 8.8g, _{CuSO 4 · 5H 2 O 0.4g,} NaB 4 O 7 · 10H 2 O 0.1g, the _{(NH 4) 6 Mo 7 O} 24 · 4H 2 O 0.05g) 1ml, agar 20 g (pH 6.5)) It was applied and DNA was introduced by the particle delivery method. Using PDS-1000 / He (Bio-Rad), particles: tungsten M-10, rupture disc: 1100 psi, distance: 3 cm. As a transformant, a strain that grows on a CD plate was selected. The transformant obtained by transforming with the plasmid pUNA-AOBGL11g was designated as the BGL11-1 strain, and the transformant obtained by transforming with the control vector pUNA was designated as the C-1 strain.

Expression of AOBGL11p by Jiuqu BGL11-1 strain or C-1 strain was inoculated on a CD plate and cultured at 30 ° C. for 7 days to form conidia. To obtain a conidia suspension, 0.1% tween80, 0.8% NaCl was added and the conidia were suspended. After filtering with Miracross (registered trademark), the saplings were collected by centrifugation, further washed with 0.1% yeast80, 0.8% NaCl, suspended in sterilized water, and used as a sprouting suspension. conidia suspension liquid medium for enzyme production (per 1L, maltose 100 g, Bacto tryptone 1g, yeast extract _{5g, NaNO 3 1g, K 2} HPO 4 0.5g, MgSO 4 · 7H 2 O 0.5g, FeSO 4 -Inoculated into 7H ₂ O 0.01 g) and shake-cultured at 30 ° C. for 2 days. Bacterial cells were collected by filtration through Miracross (registered trademark). About 0.4 g of the obtained wet cells was frozen in liquid nitrogen and ground in a mortar. The ground cells were suspended in 50 mM sodium phosphate buffer (pH 7.0), mixed well, and then centrifuged. The resulting supernatant is concentrated by ultrafiltration with Amicon® Ultra-15 50k (Merck) and replaced with 50 mM sodium phosphate buffer pH 7.0 (buffer A) containing 0.1% CHAPS. As a result, about 1 ml of a crude enzyme solution was obtained.

Measurement of protein concentration The protein concentration of the crude enzyme solution was quantified using the protein assay CBB solution (5-fold concentrated) (Nacalai Tesque). As a result, BGL11-1 crude enzyme solution was 6.46 mg / ml and C-1 crude enzyme solution was 4 mg / ml.

[Example 3]
Degradation activity of pNP-β-Glc The degradation activity of pNP-β-Glc was examined. 10 μL of crude enzyme solution, 50 μL of 0.2 M sodium phosphate buffer (pH 7.0), 50 μL of 20 mM pNP-β-Glc aqueous solution, and water were added to make a total of 200 μL, and the reaction was carried out at 37 ° C. Since the activity of the BGL11-1 crude enzyme solution was high, the crude enzyme solution diluted 100-fold with a 50 mM sodium phosphate buffer (pH 7.0) buffer containing 0.1% CHAPS was used. The change in absorbance (Δ405) at 405 nm per minute based on p-nitrophenol (pNP) released by hydrolyzing pNP-β-Glc was 0.244 in BGL11-1 crude enzyme solution, and C-1 crude enzyme. It was 0.000 in liquid. From the above, it was suggested that AOBGL11p is responsible for β-glucosidase activity.

Using pNP-β-Glc as a substrate, the optimum temperature, optimum pH, thermal stability, and pH stability of AOBGL11p were examined (FIG. 2 (FIG. 2A-2D)). The BGL11-1 crude enzyme solution used was diluted 5000-fold with buffer A (protein concentration 1.3 μg / ml).

Optimal temperature: The reaction solution is a crude enzyme solution (1.3 μg / ml) 20 μl, 0.2 M sodium phosphate buffer (pH 6.5) 100 μl, 20 mM pNP-β-Glc, and water is added to make a total of 400 μl, and the reaction is carried out. At 15, 30, and 45 minutes from the start, 100 μl was sampled and mixed with 100 μl 0.2 M sodium carbonate solution, and then the absorbance at 405 nm was measured to determine Δ405. The ratio of Δ405 when the reaction was carried out at each temperature was shown in FIG. 2A, where 45 ° C. at which Δ405 was the maximum was set to 1. From this, it was found that 45-50 ° C. is the optimum reaction temperature.

Optimal pH: The reaction solution was 20 μl of a crude enzyme solution (1.3 μg / ml), 100 μl of a 0.2 M buffer, 20 mM pNP-β-Glc, and water to make a total of 400 μl. As the buffer, a sodium acetate buffer was used when the pH was 4.0-6.0, and a sodium phosphate buffer was used when the pH was 6.0-8.0. Sampling and measurement were performed in the same manner as above, and the ratio of Δ405 to the maximum value of Δ405 when reacted at each pH is shown in FIG. 2B. From this, it was found that pH 6.0-7.0 is the optimum pH for the reaction.

Thermal stability: A crude enzyme solution (1.3 μg / ml) diluted 5000 times was held at 30 ° C., 37 ° C., 45 ° C., and 50 ° C. for 10 minutes, respectively, and then ice-cooled. The reaction solution was prepared by adding 5 μl of crude enzyme solution (1.3 μg / ml), 100 μl of 0.2 M sodium phosphate buffer (pH 6.5), 20 mM pNP-β-Glc, and water to make a total of 100 μl, and at 37 ° C. for 45 minutes. After the reaction, 100 μl of 0.2 M sodium carbonate solution was added, and the absorbance at 405 nm was measured. The ratio of the unheat-treated enzyme solution to the absorbance at 405 nm after 45 minutes at each temperature was determined and shown in FIG. 2C. It was found that AOBGL11p was stable up to 37 ° C. after 10 minutes of treatment, but was inactivated to about half by treatment at 45 ° C., and almost lost its activity by treatment at 50 ° C.

pH stability: Crude enzyme solution, pH 4.5, 5.0, 5.5, 6.0 (0.2 M acetate buffer), pH 6.0, 6.5, 7.0, 7.5, 8. It was diluted 5000 times with each buffer of 0 (0.2 M sodium phosphate buffer), kept at 37 ° C. for 1 hour, and then ice-cooled. The reaction solution was prepared by adding 5 μl of crude enzyme solution (1.3 μg / ml), 100 μl of 0.2 M sodium phosphate buffer (pH 6.5), 20 mM pNP-β-Glc, and water to make a total of 100 μl, and at 37 ° C. for 45 minutes. After the reaction, 100 μl of 0.2 M sodium carbonate solution was added, and the absorbance at 405 nm was measured. The ratio of the absorbance at each pH to the absorbance at pH 6.5, which had the highest activity, was determined and shown in FIG. 2D. AOBGL11p was found to be the most stable near pH 6.5.

[Example 4]
Cloning of AOBGL11 cDNA BGL11-1 strain was cultured in 10 ml of enzyme production medium, and bacterial cells were collected by filtration. The cells were frozen in liquid nitrogen, the cells were ground in a mortar, and then total RNA was extracted with RNeasy (QIAGEN). CDNA was synthesized by SuperScript Double-Stranded cDNA Synthesis Kit (Life Technologies). Using this as a template, PCR was performed with KOD-plus (Toyobo) using primers AOBGL11-F and AOBGL11-R. The obtained DNA fragment of about 2.52 kbp was cloned with AOBGL11 cDNA using Zero Blunt TOPO PCR Cloning Kit (Invitrogen) to obtain plasmid pCR-AOBGL11 cDNA. When the base sequence was confirmed, it was as shown in SEQ ID NO: 1, and the estimated amino acid sequence was as shown in SEQ ID NO: 2. A comparison of the genomic DNA sequence and the cDNA sequence of AOBGL11 is shown in FIG. The identity with the estimated amino acid sequence of β-glucosidase derived from the bacterial KB0549 strain belonging to the genus Paenibacillus, whose STG hydrolyzing activity has already been confirmed, was 30.3%.

[Example 5]
Production of AOBGL11p in yeast Construction of expression vector for yeast and transformation of yeast The DNA fragment of about 2.52 kbp obtained by digesting the plasmid pCR-AOBGL11 cDNA with EcoRI was used as the yeast expression vector pYE22m (Biosci. Biotech. Biochem). ., 59, 1221-1228, 1995) was inserted into the EcoRI site, and the one in which AOBGL11 was inserted in the direction of expression from the GAPDH promoter of the vector pYE22m was selected and used as pYE-AOBGL3c. As the parent strain for transformation, EH13-15 strain (trp1, MATα) of S. cerevisiae (Appl. Microbiol. Biotechnol., 30, 515-520, 1989) was used. The EH13-15 strain was transformed by the lithium acetate method using plasmids pYE22m (control) and pYE-AOBGL11 (for AOBGL11 expression), respectively. The transformants included SC-Trp (6.7 g of Yast nitrogen base w / o amino acids (DIFCO) per liter, 20 g of glucose, and amino acid powder (1.25 g of methionine sulfate, 0.6 g of arginine, 3 g of aspartic acid). Glutamic acid 3g, histidine 0.6g, leucine 1.8g, lysine 0.9g, methionine 0.6g, phenylalanine 1.5g, serine 11.25g, tyrosine 0.9g, valine 4.5g, threonine 6g, uracil 0.6g The one that grows on an agar medium (2% agar) (containing 1.3 g) was selected.

The strain obtained by transformation with the plasmid pYE22m was designated as the CY strain, and the strain obtained by transformation with the plasmid pYE-AOBGL11 was designated as the AOBGL11-Y strain. The selected CY strain and AOBGL11-Y strain were inoculated into 10 mL of SC-Trp liquid medium supplemented with 1/10 amount of 1 M potassium phosphate buffer, and cultured with shaking at 30 ° C. and 125 rpm for 2 days. .. The obtained culture was separated into culture supernatant and bacterial cells by centrifugation. The culture supernatant was concentrated to limit filtration with Amicon® Ultra-15 50 k (Merck), buffer-substituted with 50 mM sodium phosphate buffer (pH 7.0) containing 0.1% CHAPS, and about 1 ml of culture was cultured. A supernatant concentrate was obtained.

The cells were suspended in 1 ml of a 0.1% CHAPS solution in 50 mM sodium phosphate buffer (pH 7.0), the cells were crushed with glass beads, and the supernatant obtained by centrifugation was crushed. It was made into a liquid. When 20 μl of the culture supernatant concentrate or the cell disruption solution was taken, 1 μl of a 2% X-β-Glc / DMF solution was added, and the mixture was reacted at room temperature for 5 minutes, only the cell disruption solution derived from the AOBGL11-Y strain was blue. It was suggested that it had X-β-Glc activity.

Measurement of pNP-β-Glc activity The degrading activity of pNP-β-Glc was examined. 10 μL of crude enzyme solution, 50 μL of 0.2 M sodium phosphate buffer (pH 7.0), 50 μL of 20 mM pNP-β-Glc aqueous solution, and water were added to make a total of 200 μL, and the reaction was carried out at 37 ° C. Since the activity of the BGL11-1 crude enzyme solution was high, the crude enzyme solution diluted 100-fold with a 50 mM sodium phosphate buffer (pH 7.0) buffer containing 0.1% CHAPS was used. The change in absorbance (Δ405) at 405 nm per minute based on p-nitrophenol (pNP) released by hydrolysis of pNP-β-Glc was 0.068 in AOBGL11-Y crude enzyme solution, and CY crude enzyme. It was 0.000 in liquid.

[Example 6]
Sesaminol glycoside hydrolyzing activity of AOBGL11p Sesaminol triglucoside (STG) was used as a substrate. The total amount of STG was 100 μL with 50 μg / mL, 50 mM sodium phosphate buffer (pH 7.0), and 20 μL of the above BGL11-1 crude enzyme solution or a diluted solution thereof, and the mixture was reacted at 37 ° C. for 1 hour. As a control, the above C-1 crude enzyme solution was also reacted in the same manner. The reaction solution was treated at 100 ° C. for 3 minutes to stop the reaction, filtered through a Cosmos pin filter H (Nacalai Tesque), and then subjected to HPLC.

The analysis conditions for HPLC are as follows.

Column: Cosmo Seal 5C _18- AR-II 4.6 mm I. D. x250mm (Nacalai Tesque)

Mobile phase: A; 0.1% (v / v) trifluoroacetic acid, 2: 8 (v / v) acetonitrile: water B; 0.1% (v / v) trifluoroacetic acid, 8: 2 (v / v / v / v) Acetonitrile: water

B conc. 10% (0 minutes-3 minutes) → 100% 24 minutes liner
gradient, 100% (24-35 minutes)

Flow velocity: 0.7 ml / min Temperature: 40 ° C
Detection: UV 290 nm

The results are shown in FIG.

When reacted with a 10-fold diluted BGL11-1 crude enzyme solution, a part of STG of the substrate was hydrolyzed to produce SDG (1,6) and SDG (1,2), and sesaminol which is an aglycone. (Fig. 4A).
When the BGL11-1 crude enzyme solution was reacted without dilution, the STG added as a substrate was almost hydrolyzed to produce SDG (1, 2) and sesaminol, which is an aglycone (FIG. 4B). .. When reacted with a 10-fold diluted BGL11-1 crude enzyme solution, the amount of SDG (1,2) was larger than that of SDG (1,6), and the BGL11-1 crude enzyme solution was reacted without being diluted. In this case, since SDG (1,6) is not detected and only SDG (1,2) is detected, AOBGL11p is β-1, compared with β-1,2 binding in the branched sugar chain of STG. It was suggested that it works well on 6 bonds.
From the above results, it is shown that AOBGL11p is a mode in which β-1,6 bonds are preferentially hydrolyzed over β-1,2 bonds and finally hydrolyzed to sesaminol, which is an aglycone. Was done. It was also shown that it is possible to control the progress of hydrolysis of sesaminol glycosides by adjusting the concentration of the enzyme solution. The result of the reaction between the C-1 crude enzyme solution and STG is shown in FIG. 4C, and this result indicates that the C-1 crude enzyme solution does not promote the hydrolysis of STG.

The present invention provides a method for producing sesaminol and / or sesaminol glycosides by hydrolyzing sesaminol triglucoside using AOBGL11p, which is a glucoside hydrase derived from Jiuqu.

Claims (15)

A protein selected from the group consisting of the following (a) to (c) is reacted with a substrate sesaminol glycoside having at least one glucoside bond to form at least one glucoside bond of the substrate sesaminol glycoside. A method for producing a produced sesaminol or a produced sesaminol glycoside, which comprises a step of hydrolyzing.
(A) A protein consisting of the amino acid sequence of SEQ ID NO: 2;
(B) A substrate sesaminol glycoside consisting of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2 and having at least one glucosidic bond. A protein that has the activity of hydrolyzing the glycosidic bond of
(C) Activity of hydrolyzing the glucosidic bond of the substrate sesaminol glycoside having 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 and having at least one glucosidic bond. Protein with The substrate sesaminol glycosides are sesaminol 2'-O-β-D-glucopyranoside (sesaminol monoglucoside, SMG) and 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 (sesame) Nord (1-6) diglucoside, SDG (1,6)), and sesaminol 2'-O-β-D-glucopyranosyl (1-2) -O- (-β-D-glucopyranosyl (1-2)) The method according to claim 1, wherein the method is any one selected from the group consisting of β-D-glucopyranoside (sesaminol triglucoside, STG). The method according to claim 1 or 2, wherein the substrate sesaminol glycoside is STG. Claim 1 or 2, wherein the produced sesaminol or produced sesaminol glycoside is any one or more selected from the group consisting of SDG (1,6), SDG (1,2) and sesaminol. The method described. The at least one glucosidic bond is a glucosidic bond between glucose and aglycon bound to the sesaminol 2'position, a β-1,6-glucoside bond of gentiobiose bound to the sesaminol 2'position, and a sesaminol 2'position. Either the β-1,2 bond of sophorose bound to, or the β-1,6-glucosidic bond or β-1,2 bond of the branched trisaccharide bound to sesaminol at the 2'position. The method according to any one of claims 1 to 4, which is one. An enzyme agent derived from a non-human transformed cell into which a polynucleotide selected from the group consisting of the following (a) to ( d) has been introduced into a host cell, a substrate sesaminol glycoside having at least one glucosidic bond. A method for producing a produced sesaminol or produced sesaminol glycoside, which comprises a step of hydrolyzing at least one glucoside bond of the substrate sesaminol glycoside in contact with the body.
(A) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;
(B) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(C) A substrate sesaminol glycoside consisting of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2 and having at least one glucosidic bond. A polynucleotide encoding a protein that has the activity of hydrolyzing the glycosidic bond of
(D) Activity of hydrolyzing the glucosidic bond of a substrate sesaminol glycoside having an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 and having at least one glucoside bond. polynucleotide which encodes a protein having a
The method of claim 6, wherein the polynucleotide is inserted into an expression vector. The method according to claim 6 or 7, wherein the transformed cells are selected from the group consisting of transformed plants, transformed Escherichia coli, transformed bacteria, transformed yeast, and transformed filamentous fungi. The substrate sesaminol glycosides are sesaminol 2'-O-β-D-glucopyranoside (sesaminol monoglucoside, SMG) and 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 (sesame) Nord (1-6) diglucoside, SDG (1,6)), and sesaminol 2'-O-β-D-glucopyranosyl (1-2) -O- (-β-D-glucopyranosyl (1-2)) The method according to any one of claims 6 to 8, which is any one selected from the group consisting of β-D-glucopyranoside (sesaminol triglucoside, STG). Claims 6 to 9, wherein the produced sesaminol or produced sesaminol glycoside is any one or more selected from the group consisting of SDG (1,6), SDG (1,2) and sesaminol. The method according to any one item. The at least one glucosidic bond is a glucosidic bond between glucose and aglycon bound to the sesaminol 2'position, a β-1,6-glucoside bond of gentiobiose bound to the sesaminol 2'position, and a sesaminol 2'position. Either the β-1,2 bond of sophorose bound to, or the β-1,6-glucosidic bond or β-1,2 bond of the branched trisaccharide bound to sesaminol at the 2'position. The method according to any one of claims 6 to 10, which is one. A method for producing a produced sesaminol or a produced sesaminol glycoside, which comprises culturing a non-human transformant into which a polynucleotide selected from the group consisting of the following (a) to ( d) has been introduced.
(A) A polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1;
(B) A polynucleotide encoding a protein consisting of the amino acid sequence of SEQ ID NO: 2;
(C) A substrate sesaminol glycoside consisting of an amino acid sequence in which 1 to 83 amino acids are deleted, substituted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 2 and having at least one glucosidic bond. A polynucleotide encoding a protein that has the activity of hydrolyzing the glycosidic bond of
(D) Activity of hydrolyzing the glucosidic bond of a substrate sesaminol glycoside having an amino acid sequence having 90% or more sequence identity with respect to the amino acid sequence of SEQ ID NO: 2 and having at least one glucoside bond. polynucleotide which encodes a protein having a The method of claim 10, wherein the polynucleotide is inserted into an expression vector. The method according to claim 12 or 13, wherein the transformant is selected from the group consisting of transformed plants, transformed Escherichia coli, transformed bacteria, transformed yeast, and transformed filamentous fungi. The at least one glucosidic bond is a glucosidic bond between glucose and aglycon bound to the sesaminol 2'position, a β-1,6-glucoside bond of gentiobiose bound to the sesaminol 2'position, and a sesaminol 2'position. Either the β-1,2 bond of sophorose bound to, or the β-1,6-glucosidic bond or β-1,2 bond of the branched trisaccharide bound to sesaminol at the 2'position. The method according to any one of claims 12 to 14, which is one.

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