Method for producing β-glucosyl glycoside and transformant

Abstract

To provide a novel method for producing β-glucosyl glycosides, and a novel transformant suitable for use in the production of β-glucosyl glycosides.SOLUTION: A method for producing β-glucosyl glycosides according to the present invention comprises subjecting a system comprising a substrate compound having a hydroxyl group and a glucose donor having a β-glycosidic bond to a microorganism such as Ensifer adhaerens or a β-glycosylation enzyme derived from the microorganism, and then recovering the β-glucosyl glycoside of the substrate compound.SELECTED DRAWING: Figure 2

Description

Technical Field

[0001] The present invention relates to a method for producing β-glucosyl glycosides and a transformant.

Background Art

[0002] In order to improve the physical properties (such as water solubility and stability) of various compounds such as polyphenols and terpene alcohols, research on glycosylation of these compounds has been actively conducted. In particular, many of the naturally occurring glycosides are β-glycosides such as β-glucosyl glycosides, and development of an efficient method for producing β-glycosides is desired.

[0003] Conventionally, as a method for producing β-glycosides, a method using a transglycosylation reaction by β-glucosidase is known. For example, in Non-Patent Document 1, β-glucosidase derived from almond or Trichoderma reesei was allowed to act on a system containing geraniol as a substrate compound and cellobiose as a sugar donor, and it was reported that β-glucosyl glycoside of geraniol was obtained.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, conventionally known β-glucosidases such as almond-derived β-glucosidase mainly promote hydrolysis reactions, and the yield of β-glucosyl glycosides has been hardly sufficient.

[0006] Therefore, an object of the present invention is to provide a method for producing a novel β-glucosyl glycoside and a novel transformant suitably used for producing a β-glucosyl glycoside.

Means for Solving the Problems

[0007] Specific means for solving the above problems include the following embodiments. <1> A method for producing a β-glucosyl glycoside, which comprises allowing any one of the microorganisms (1) to (24) below or a β-glycosylating enzyme derived from the microorganism to act on a system containing a substrate compound having a hydroxyl group and a glucose donor having a β-glycosidic bond, and recovering the β-glucosyl glycoside of the substrate compound. (1) Ensifer adhaerens (2) Shinella yambaruensis (3) Shinella granuli (4) Shinella zoogloeoides (5) Aspergillus luchuensis (6) Aspergillus tubingensis (7) Talaromyces albobiverticillius (8) Penicillium janthinellum (9) Penicillium adametzii (10) Phaeosphaeria oryzae (11) Pyrenophora dictyoides (12) Marasmiellus mesosporus (13) Omphalotus guepiniiformis (14) Agrocybe cylindracea (15) Torula dematia (16) Grifola frondosa (17) Aureobasidium pullulans (18) Pichia farinosa (19) Cyberlindnera saturnus (20) Papiliotrema aurea (21) Daedalea dickinsii (22) Daedalea serialis (23) Lentinula edodes (24) Coprinopsis cinerea

[0008] <2> A method for producing a β - glucosyl glycoside, which comprises allowing a transformant into which a gene encoding a β - glucosidase derived from any one of the following microorganisms (1) to (28) is introduced to act on a system containing a substrate compound having a hydroxyl group and a glucose donor having a β - glycosidic bond, and recovering the β - glucosyl glycoside of the substrate compound. (1) Ensifer adhaerens (2) Shinella yambaruensis (3) Shinella granuli (4) Shinella zoogloeoides (5) Aspergillus luchuensis (6) Aspergillus tubingensis (7) Talaromyces albobiverticillius (8) Penicillium janthinellum (9) Penicillium adametzii (10) Phaeosphaeria oryzae (11) Pyrenophora dictyoides (12) Marasmiellus mesosporus (13) Omphalotus guepiniiformis (14) Agrocybe cylindracea (15) Torula dematia (16) Grifola frondosa (17) Aureobasidium pullulans (18) Pichia farinosa (19) Cyberlindnera saturnus (20) Papiliotrema aurea (21) Daedalea dickinsii (22) Daedalea serialis (23) Lentinula edodes (24) *Coprinopsis cinerea* (25) *Rhizobium radiobacter* (26) *Rhizobium pusense* (27) *Rhizobium daejeonense* (28) *Rhizobium paknamense*

[0009] <3> A method for producing a β-glucosyl glycoside, comprising recovering the β-glucosyl glycoside of the substrate compound by introducing a gene encoding any one of the following proteins (a) to (c) into a system containing a substrate compound having a hydroxyl group and a glucose donor having a β-glycosidic bond, or by allowing any one of the following proteins (a) to (c) to act thereon. (a) A protein consisting of the amino acid sequence of SEQ ID NO: 1 or 2. (b) A protein consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, or added in the amino acid sequence of (a) and having β-glucosidase activity. (c) A protein having 90% or more sequence identity with the amino acid sequence of (a) and having β-glucosidase activity.

[0010] <4> The method for producing a β-glucosyl glycoside according to any one of <1> to <3>, wherein the glucose donor is cellobiose.

[0011] <5> A transformant into which a gene encoding any one of the following proteins (a) to (c) has been introduced. (a) A protein consisting of the amino acid sequence of SEQ ID NO: 1 or 2. (b) A protein consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, or added in the amino acid sequence of (a) and having β-glucosidase activity. (c) A protein having a sequence identity of 90% or more with the amino acid sequence of (a) and having β-glucosidase activity.

Advantages of the Invention

[0012] According to the present invention, it is possible to provide a novel method for producing a β-glucosyl glycoside and a novel transformant suitably used in producing a β-glucosyl glycoside.

Brief Description of the Drawings

[0013]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0014] Hereinafter, specific embodiments to which the present invention is applied will be described. In this specification, the numerical range indicated using "~" indicates a range including the numerical values described before and after "~".

[0015] <Method for Producing β-Glucosyl Glycoside According to the First Aspect> The method for producing a β - glucosyl glycoside according to the first aspect (hereinafter simply referred to as "the production method according to the first aspect") involves allowing any one of the microorganisms (1) to (24) described below or a β - glycosylating enzyme derived from the microorganism to act on a system containing a substrate compound having a hydroxyl group and a glucose donor having a β - glycosidic bond, and recovering the β - glucosyl glycoside of the substrate compound.

[0016] [Microorganism or β - glycosylating enzyme derived from the microorganism] In the production method according to the first aspect, any one of the microorganisms (1) to (24) described below or a β - glycosylating enzyme derived from the microorganism is used. (1) Ensifer adhaerens (2) Shinella yambaruensis (3) Shinella granuli (4) Shinella zoogloeoides (5) Aspergillus luchuensis (6) Aspergillus tubingensis (7) Talaromyces albobiverticillius (8) Penicillium janthinellum (9) Penicillium adametzii (10) Phaeosphaeria oryzae (11) Pyrenophora dictyoides (12) Marasmiellus mesosporus (13) Omphalotus guepiniiformis (14) Agrocybe cylindracea (15) Torula dematia (16) Grifola frondosa (17) Aureobasidium pullulans (18) Pichia farinosa (19) Cyberlindnera saturnus (20) Papiliotrema aurea (21) Daedalea dickinsii (22) Daedalea serialis (23) Lentinula edodes (24) Coprinopsis cinerea

[0017] The β-glycoside hydrolase derived from the microorganisms (1) to (24) above is, for example, β-glucosidase. This β-glycoside hydrolase is characterized by having a higher synthetic activity of β-glucosyl glycoside (β-glucosyl transferase activity), particularly the synthetic activity of β-monoglucosyl glycoside, compared to conventionally known β-glucosidases such as almond-derived β-glucosidase. Therefore, by using the microorganisms (1) to (24) above or the β-glycoside hydrolase derived from the microorganisms, it is possible to obtain β-glucosyl glycoside (particularly, β-monoglucosyl glycoside) of a substrate compound in a high yield.

[0018] Specific strains of the microorganisms (1) to (24) above include, for example, Enterococcus adhaerens NBRC 100388, Sinella yambaruensis NBRC 102122, Sinella granulii JCM 13254, Sinella zoochrooides JCM 20728, Aspergillus luchuensis NBRC 4033, Aspergillus tubingensis NBRC 4050, Aspergillus tubingensis JCM 5697, Talaromyces albobiverticillius NBRC 6580, Penicillium janthinellum NBRC 4651, Penicillium adametzioi NBRC 7680, Phaeosphaeria oryzae NBRC 33076, Pyrenophora dictyoides NBRC 7370, Marasmiellus mesosporus NBRC 105515, Omphalotus guepiniformis NBRC 6992, Agrocybe cylindracea NBRC 9075, Torula dematia NBRC 6212, Grifola frondosa NBRC 7040, Aureobasidium pullulans NBRC 4465, Pichia farinosa NBRC 0193, Sibirindonesia saturnus NBRC 0992, Sibirindonesia saturnus NBRC 0941, Papiliotrema aurea NBRC 0372, Daedalea dickinsii NBRC 31163, Daedalea cerealis NBRC 9286, LentinuIa edodes NBRC 8340, Coprinopsis cinerea NBRC 30628, etc. These strains can be easily obtained from the Microbial Materials Development Laboratory (JCM) of the RIKEN BioResource Center or the Biotechnology Center of the National Institute of Technology and Evaluation (NBRC).

[0019] In the production method according to the first aspect, the microorganisms (1) to (24) themselves may be used, or a processed product obtained by treating a culture of the microorganisms (for example, culture supernatant) may be used, or a β-glycosidase separated and purified from the culture may be used. The separation and purification of β-glycosidase can be carried out by combining the disrupted cells or culture supernatant with ordinary enzyme purification treatment means such as ammonium sulfate precipitation, ion exchange column chromatography, chelate affinity chromatography, gel filtration column chromatography, etc.

[0020] [Substrate compound] The substrate compound is not particularly limited as long as it is a compound having one or more hydroxyl groups that can be glycosylated by the β-glycosidase derived from the microorganisms (1) to (24) above, and it may be a compound having an alcoholic hydroxyl group or a compound having a phenolic hydroxyl group.

[0021] Examples of the compound having an alcoholic hydroxyl group include terpene alcohols such as 3-methyl-2-buten-1-ol, 3-methyl-3-buten-2-ol, nerol, geraniol, linalool, terpineol, citronellol, nerolidol, farnesol, geranylgeraniol, phytol; aliphatic alcohols such as methanol, ethanol, 1-propanol, 1-butanol, 2-butanol, 1-pentanol, 1-hexanol, 1-octanol, benzyl alcohol, 2-phenylethanol, etc., which may be substituted with an aromatic group; and the like.

[0022] Examples of the compound having a phenolic hydroxyl group include phenols such as phenol, hydroquinone, pyrocatechol, salicyl alcohol, phloretin, resveratrol, curcumin, and tetrahydrocurcumin; coumarins such as esculetin; flavonoids such as apigenin, daidzein, quercetin, hesperetin, and naringenin; chalcones such as chalcone; anthocyanidins such as pelargonidin, cyanidin, and delphinidin; anthraquinones such as alizarin; indoles such as indoxyl; alkaloids such as dopamine and adrenaline; and the like.

[0023] [Glucose donor] The glucose donor is not particularly limited as long as it contains a glucose residue bonded by a β-glycosidic bond, and cellobiose, gentiobiose, etc. are preferably exemplified.

[0024] [Production method] In the production method according to the first aspect, the above-mentioned microorganisms (1) to (24) or a β-glycosidase derived from the microorganisms is allowed to act on a system containing a substrate compound and a glucose donor to produce a β-glucosyl glycoside of the substrate compound. For example, a substrate compound and a glucose donor are added to the above-mentioned microorganisms (1) to (24) or a treated product (e.g., culture supernatant) obtained by treating a culture of the microorganisms, and the obtained enzyme reaction solution is incubated to produce a β-glucosyl glycoside of the substrate compound.

[0025] The concentration of the substrate compound in the enzyme reaction solution is usually 1 to 50 g / L, preferably 3 to 10 g / L. The concentration (g / L) of the glucose donor is preferably in the range of 0.5 to 30 times that of the substrate compound.

[0026] The concentration of the β-glycosidase in the enzyme reaction solution is preferably such that the reaction can be completed in about 1 to 100 hours. Usually, it is 0.1 to 100 U per 1 g of the glucose donor, preferably 0.2 to 50 U. Note that 1 U represents the amount of enzyme that allows a chemical reaction of 1 μmol of substrate to proceed per minute.

[0027] The pH and temperature of the enzymatic reaction solution may be any conditions under which the β-glycosidase acts sufficiently, and are usually pH 5 to 10 and temperature 15 to 50°C.

[0028] Since the solubility of the substrate compound and its β-glucosyl glycoside in water is different, it is possible to separate them by an extraction operation. At this time, since the β-glucosyl glycoside of the substrate compound dissolves in a more hydrophilic solvent, a high-purity β-glucosyl glycoside can be recovered by further purifying the β-glucosyl glycoside dissolved in this solvent.

[0029] <Method for producing β-glucosyl glycoside according to the second aspect> The method for producing a β-glucosyl glycoside according to the second aspect (hereinafter, simply referred to as "the production method according to the second aspect") is to act on a system containing a substrate compound having a hydroxyl group and a glucose donor having a β-glycoside bond with a transformant into which a gene encoding a β-glycosidase derived from any one of the following microorganisms (1) to (28) is introduced, and recover the β-glucosyl glycoside of the substrate compound.

[0030] [Transformant] In the production method according to the second aspect, a transformant into which a gene encoding a β-glycosidase derived from any one of the following microorganisms (1) to (28) is introduced is used. (1) Ensifer adhaerens (2) Shinella yambaruensis (3) Shinella granuli (4) Shinella zoogloeoides (5) Aspergillus luchuensis (6) Aspergillus tubingensis (7) Talaromyces albobiverticillius (8) Penicillium janthinellum (9) Penicillium adametzii (10) Phaeosphaeria oryzae (11) Pyrenophora dictyoides (12) Marasmiellus mesosporus (13) Omphalotus guepiniiformis (14) Agrocybe cylindracea (15) Torula dematia (16) Grifola frondosa (17) Aureobasidium pullulans (18) Pichia farinosa (19) Cyberlindnera saturnus (20) Papiliotrema aurea (21) Daedalea dickinsii (22) Daedalea serialis (23) Lentinula edodes (24) Coprinopsis cinerea (25) Rhizobium radiobacter (26) Rhizobium pusense (27) Rhizobium daejeonense (28) Rhizobium paknamense

[0031] The β - glycoside hydrolase derived from the microorganisms (1) to (28) above is, for example, β - glucosidase. This β - glycoside hydrolase has the characteristic that its synthetic activity of β - glucosyl glycoside (β - glucosyltransferase activity), particularly the synthetic activity of β - monoglucosyl glycoside, is higher than that of conventionally known β - glucosidases such as almond - derived β - glucosidase. Therefore, by using a transformant into which a gene encoding the β - glycoside hydrolase derived from the microorganisms (1) to (28) above is introduced, it is possible to obtain β - glucosyl glycosides (particularly, β - monoglucosyl glycosides) of substrate compounds in high yields.

[0032] Specific strains of the microorganisms (1) to (24) above include the strains exemplified in the production method according to the first aspect. Also, specific strains of the microorganisms (25) to (28) above include Rhizobium radiobacter JCM 20371 strain, Rhizobium pusense JCM 10269 strain, Rhizobium daejeonense JCM 21505 strain, Rhizobium paknamense NBRC 109338 strain, etc. These strains can be easily obtained from the Microbial Materials Development Laboratory of the RIKEN BioResource Center (JCM) or the Biotechnology Center of the National Institute of Technology and Evaluation (NBRC).

[0033] A transformant can be prepared by transforming a host cell such as Escherichia coli with a gene encoding the β - glycoside hydrolase derived from the microorganisms (1) to (28) above so that the β - glycoside hydrolase can be expressed.

[0034] Generally, it is known that a protein can have the same activity even if one or several amino acid residues in its amino acid sequence are substituted, deleted, or added. Therefore, the β-glycosidase expressed by the transformant may be a modified β-glycosidase in which one or several amino acid residues are substituted, deleted, or added in the amino acid sequence of the wild-type β-glycosidase. The sequence identity between the amino acid sequence of the wild-type β-glycosidase and that of the modified β-glycosidase is usually 90% or more, and may be 92% or more, 94% or more, 96% or more, or 98% or more. Here, "sequence identity" means the consistency between sequences when two amino acid sequences are aligned, and can be calculated using, for example, the BLAST program (www.ncbi.nlm.nih.gov / Blast / cgi).

[0035] [Substrate compound and glucose donor] As the substrate compound and the glucose donor, the same components as those in the production method according to the first embodiment can be used.

[0036] [Production method] In the production method according to the second embodiment, a transformant into which a gene encoding β-glycosidase derived from the above-mentioned microorganisms (1) to (28) is introduced is allowed to act on a system containing a substrate compound and a glucose donor to produce a β-glucosyl glycoside of the substrate compound. For example, a substrate compound and a glucose donor are added to the transformant, and the obtained enzyme reaction solution is incubated to produce a β-glucosyl glycoside of the substrate compound.

[0037] The concentration of the substrate compound in the enzyme reaction solution is usually 1 to 50 g / L, preferably 3 to 10 g / L. Also, the concentration (g / L) of the glucose donor is preferably in the range of 0.5 to 30 times that of the substrate compound.

[0038] The concentration of β-glycosidase in the enzyme reaction solution is preferably such that the reaction can be completed in about 1 to 100 hours, and is usually 0.1 to 100 U, preferably 0.2 to 50 U, per 1 g of the glucose donor. Here, 1 U represents the amount of enzyme that allows the chemical reaction of 1 μmol of substrate to proceed per minute.

[0039] The pH and temperature of the enzyme reaction solution may be any conditions under which β-glycosidase can sufficiently act, and are usually pH 5 to 10 and temperature 15 to 50°C.

[0040] Since the solubility of the substrate compound and its β-glucosyl glycoside in water is different, they can be separated by an extraction operation. At this time, since the β-glucosyl glycoside of the substrate compound dissolves in a more hydrophilic solvent, a high-purity β-glucosyl glycoside can be recovered by further purifying the β-glucosyl glycoside dissolved in this solvent.

[0041] <Method for producing β-glucosyl glycoside according to the third aspect> The method for producing a β-glucosyl glycoside according to the third aspect (hereinafter, simply referred to as "the production method according to the third aspect") includes causing a transformant into which a gene encoding a specific protein described below is introduced or a specific protein to act on a system containing a substrate compound having a hydroxyl group and a glucose donor having a β-glycoside bond, and recovering the β-glucosyl glycoside of the substrate compound.

[0042] [Transformant or protein] In the production method according to the third aspect, a transformant into which a gene encoding any of the following proteins (a) to (c) is introduced or any of the following proteins (a) to (c) is used.

[0043] (a) A protein consisting of the amino acid sequences of SEQ ID NOs: 1 to 4. (b) A protein consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, or added in the amino acid sequence of (a) above, and having β-glucosidase activity. (c) A protein having a sequence identity of 90% or more with the amino acid sequence of (a) above and having β-glucosidase activity.

[0044] The proteins (β-glucosidases) of (a) to (c) above are characterized by having a higher synthetic activity of β-glucosyl glycosides (β-glucosyltransferase activity), particularly the synthetic activity of β-monoglucosyl glycosides, compared to conventionally known β-glucosidases such as almond-derived β-glucosidase. Therefore, by using a transformant into which the gene encoding the protein of (a) to (c) above has been introduced or the protein of (a) to (c) above, it is possible to obtain β-glucosyl glycosides (particularly, β-monoglucosyl glycosides) of a substrate compound in a high yield.

[0045] The protein consisting of the amino acid sequence of SEQ ID NO: 1 in (a) above is a protein known as β-glucosidase produced by Rhizobium pusense JCM 10269 strain (GenBank accession protein ID: WP_077986965). The protein consisting of the amino acid sequence of SEQ ID NO: 2 is a protein known as β-glucosidase produced by Ensifer adherens NBRC 100388 strain (GenBank accession protein ID: WP_034787007). The protein consisting of the amino acid sequence of SEQ ID NO: 3 is a protein known as β-glucosidase produced by Sinorhizobium yambaruensis NBRC 102122 strain (GenBank accession protein ID: WP_244768214). The protein consisting of the amino acid sequence of SEQ ID NO: 4 is a protein known as β-glucosidase produced by Sinorhizobium granuli JCM 13254 strain (GenBank accession protein ID: WP_133033574).

[0046] The protein of (b) above consists of an amino acid sequence in which one or several amino acid residues are substituted, deleted, or added in the amino acid sequence of (a) above, and has β-glucosidase activity. The positions where amino acid residues are substituted, deleted, or added are not particularly limited as long as the β-glucosidase activity is maintained. Also, the number of amino acid residues to be substituted, deleted, or added is not particularly limited as long as the β-glucosidase activity is maintained. The number of amino acid residues to be substituted, deleted, or added may be, for example, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, or 1 to 5.

[0047] When substituting any amino acid residue with another amino acid residue, it is usually preferable that the properties of the amino acid side chain are conserved before and after substitution. When classifying amino acids according to the properties of the amino acid side chain, for example, hydrophilic amino acids (D, E, K, R, H, S, T, N, Q); hydrophobic amino acids (A, G, V, I, L, F, Y, W, M, C, P); acidic amino acids (D, E); basic amino acids (K, R, H); amino acids with aliphatic side chains (A, G, V, I, L); amino acids with aromatic-containing side chains (F, Y, W); amino acids with sulfur atom-containing side chains (M, C); etc. can be classified (the alphabets in parentheses indicate the single-letter notation of amino acids).

[0048] The protein of (c) above has a sequence identity of 90% or more with the amino acid sequence of (a) above and has β-glucosidase activity. The sequence identity with the amino acid sequence of (a) above may be 92% or more, 94% or more, 96% or more, or 98% or more.

[0049] A transformant can be prepared by transforming a host cell such as Escherichia coli with the gene encoding the protein of (a) to (c) above so that the protein of (a) to (c) above can be expressed.

[0050] In the production method according to the third aspect, the above-mentioned transformant itself may be used, or a processed product (for example, culture supernatant) obtained by treating the culture of the transformant may be used, or the proteins (a) to (c) separated and purified from the culture may be used. The separation and purification of the proteins (a) to (c) can be carried out by combining conventional enzyme purification treatment means such as cell disruption products or culture supernatants with ammonium sulfate precipitation, ion exchange column chromatography, chelate affinity chromatography, gel filtration column chromatography, etc.

[0051] [Substrate compound and glucose donor] As the substrate compound and glucose donor, the same components as those in the production method according to the first embodiment can be used.

[0052] [Production method] In the production method according to the third aspect, a transformant into which a gene encoding the proteins (a) to (c) is introduced or the proteins (a) to (c) are allowed to act on a system containing a substrate compound and a glucose donor to produce a β-glucosyl glycoside of the substrate compound. For example, a substrate compound and a glucose donor are added to the transformant or a processed product (for example, culture supernatant) obtained by treating the transformant of the microorganism, and the resulting enzyme reaction solution is incubated to produce a β-glucosyl glycoside of the substrate compound.

[0053] The concentration of the substrate compound in the enzyme reaction solution is usually 1 to 50 g / L, preferably 3 to 10 g / L. Also, the concentration (g / L) of the glucose donor is preferably in the range of 0.5 to 30 times that of the substrate compound.

[0054] The concentration of the proteins (a) to (c) in the enzyme reaction solution is preferably such that the reaction can be completed in about 1 to 100 hours, and is usually 0.1 to 100 U per 1 g of the glucose donor, preferably 0.2 to 50 U. Note that 1 U represents the amount of enzyme that promotes a chemical reaction of 1 μmol of substrate per minute.

[0055] The pH and temperature of the enzymatic reaction solution may be any conditions under which the proteins in the above (a) to (c) can sufficiently act, and are usually pH 5 to 10 and temperature 15 to 50 °C.

[0056] Since the solubility of the substrate compound and its β-glucosyl glycoside in water is different, they can be separated by an extraction operation. At this time, since the β-glucosyl glycoside of the substrate compound dissolves in a more hydrophilic solvent, a high-purity β-glucosyl glycoside can be recovered by further purifying the β-glucosyl glycoside dissolved in this solvent.

Examples

[0057] Hereinafter, examples of the present invention will be described, but the scope of the present invention is not limited to these examples.

[0058] <Test Example 1: Glycosylation reaction of 2-phenylethanol by various microorganisms> In Test Example 1, eight types of microorganisms that produce β-glycosylating enzyme were used, namely Ensifer adhaerens NBRC 100388 strain, Sinorhizobium yambaruense NBRC 102122 strain, Sinorhizobium granuli JCM 13254 strain, Sinorhizobium zoooglueoides JCM 20728 strain, Rhizobium radiobacter JCM 20371 strain, Rhizobium pusense JCM 10269 strain, Rhizobium daejeonense JCM 21505 strain, and Rhizobium paknamense NBRC 109338 strain.

[0059] One platinum loop of each microorganism was inoculated into a 200 mL Erlenmeyer flask containing 50 mL of the liquid medium shown in Table 1 below adjusted to pH 7.0, and cultured at 30 °C and 160 rpm for 48 hours. 0.75 mL of the obtained culture solution was dispensed into a 2 mL microtube, centrifuged at 4 °C and 15,000 rpm for 5 minutes, and 0.5 mL of phosphate buffer (20 mM KPB, pH 7.0) was added to the obtained cells for washing. Centrifugation was performed again under the same conditions as above, and the supernatant was removed to prepare washed cells of each microorganism.

[0060]

Table 1

[0061] To the washed cells of each microorganism, 40 mL of a phosphate buffer (20 mM KPB, pH 7.0) containing 7.0 g / L of 2-phenylethanol and 100 g / L of cellobiose was added as a reaction solution, and the reaction was carried out at 30 °C and 190 rpm for 96 hours in an incubator. After adding methanol to stop the reaction, centrifugation was performed, and the supernatant was collected. Then, the supernatant was analyzed by high-performance liquid chromatography (HPLC), and the accumulation amount of the glycoside (2-phenylethanol-β-D-glucopyranoside) was quantified. Specifically, 2-phenylethanol-β-D-glucopyranoside whose structure was determined by nuclear magnetic resonance (NMR) analysis was used as a standard, calibration curves were prepared by analyzing standard solutions of various concentrations by HPLC, and the accumulation amount of the glycoside was quantified from the area value of the peak showing the same retention time as the standard. The results are shown in Table 2 below. -HPLC Analysis Conditions- Column: COSMOSIL 5C 18 -AR-II (Nacalai Tesque) Column size: 4.6 × 250 mm Eluent: Acetonitrile / water = 1 / 10, 0.1% formic acid Flow rate: 1.0 mL / min Temperature of column and spectroscope: 40 °C Wavelength: λ = 215 nm

[0062]

Table 2

[0063] For reference, the accumulation amount of glycosides when using almond-derived β-glucosidase was also quantified. To 1.0 mg of almond-derived β-glucosidase, 0.5 mL of a phosphate buffer (20 mM KPB, pH 7.0) containing 7.0 g / L of 2-phenylethanol and 100 g / L of cellobiose was added as a reaction solution, and the mixture was incubated at 40 °C for 24 hours while shaking at 1200 strokes / min. The obtained reaction solution was centrifuged at 20 °C and 10,000 rpm for 1 minute, and the supernatant was recovered. Then, the supernatant was analyzed by HPLC to quantify the accumulation amount of the glycoside (2-phenylethanol-β-D-glucopyranoside). As a result, the production of 0.34 g / L of the glycoside (2-phenylethanol-β-D-glucopyranoside) was confirmed.

[0064] <Test Example 2: Preparation of Transformants> Rhizobium pusense JCM 10269 T The β-glucosidase gene (RpG3 gene) of the strain and the β-glucosidase gene (EaG3 gene) of the Ensifer adherens NBRC 100388 strain were purchased from Genbank. For the RpG3 gene (GenBank accession protein ID: WP_077986965), primers of SEQ ID NOs: 5 and 6 were used, and for the EaG3 gene (GenBank accession protein ID: WP_034787007), primers of SEQ ID NOs: 7 and 8 were used for PCR amplification, respectively. (RpG3 gene) Forward primer: 5’-AAACATATGACCGATCCCAAAACGCTCGCAGCC-3’ (SEQ ID NO: 5) Reverse primer: 5’-AAACTCGAGTCACCCCTTCACCACCCCATGG-3’ (SEQ ID NO: 6) (EaG3 gene) Forward primer: 5’-AAACATATGACCGATCCCAAGATCCTGGCAGAGC-3’ (SEQ ID NO: 7) Reverse primer: 5’-AAACTCGAGTCAGATGTGGTTGCCCTTCGGGAA-3’ (SEQ ID NO: 8)

[0065] PCR was performed using Takara Ex Taq DNA polymerase (Takara Bio). After incubation at 94°C for 1 minute, 30 cycles were carried out with cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute, followed by incubation at 72°C for 3 minutes. The expression vector pET-22b for E. coli was treated with restriction enzymes NdeI and XhoI, purified by gel electrophoresis, and then mixed with the PCR product treated with restriction enzymes NdeI and XhoI and purified by gel electrophoresis. Ligation was performed using DNA Ligation Kit (Mighty Mix; Takara Bio). Then, the obtained plasmids (pET-22b / RpG3 and pET-22b / EaG3) were transformed into E. coli (BL-21 (DE3); Merck).

[0066] <Test Example 3: Expression and glycosylation reaction of recombinant RpG3> Recombinant E. coli into which the RpG3 gene was introduced was inoculated into LB medium (50 mL) containing 50 μg / mL ampicillin and cultured with shaking at 37°C and 180 rpm for about 3 hours until the OD 600 of the culture reached 0.6. Gene expression was induced by adding isopropylthiogalactoside (IPTG) at a final concentration of 0.1 mmol / L. Furthermore, the recombinant E. coli was cultured at 18°C for 1 day. 10.0 mL of the obtained culture was dispensed into a 10 mL microtube and centrifuged at 4°C and 15,000 rpm for 5 minutes. 5.0 mL of phosphate buffer (20 mM KPB, pH 7.0) was added to the obtained bacterial cells for washing. Centrifugation was performed again under the same conditions as above, and the supernatant was removed to prepare washed bacterial cells.

[0067] To the washed cells in the microtube, 5.0 mL of a phosphate buffer solution (20 mM KPB, pH 7.0) containing 7.0 g / L 2-phenylethanol and 100 g / L cellobiose was added as a reaction solution, and the reaction was carried out under conditions of 40° C. and 190 rpm, and sampling was carried out over time. 0.5 mL of each sampling solution was added to 0.5 mL of methanol to stop the reaction, followed by centrifugation, and the supernatant was analyzed by HPLC in the same manner as in Test Example 1.

[0068] An example of a chromatogram is shown in Figure 1. A peak for the target glycoside, 2-phenylethanol-β-D-glucopyranoside, was confirmed at a retention time of 31 minutes, and a peak for the substrate compound, 2-phenylethanol, was confirmed at a retention time of 48 minutes. Three peaks (P2, P3, and P4) were also obtained at retention times of 21 minutes, 33 minutes, and 38 minutes. The reaction solution was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the product at a retention time of 31 minutes had a mass-to-charge ratio of 329 m / z, which matched the molecular weight of the target glycoside. The mass-to-charge ratios of the by-products P2, P3, and P4 were all 490 m / z, so these were predicted to be diglucosides (2-phenylethanol-diglucoside) in which one glucose was further bound to the target glycoside. Based on the properties of β-glucosidase, it is speculated that the diglucosides P2, P3, and P4 are β-1,3, β-1,4, and β-1,6 bonds, respectively.

[0069] The time course of 2-phenylethanol and glycosides is shown in Figure 2. As there were no standards for P2, P3, and P4, the concentrations were calculated using the calibration curve of 2-phenylethanol-β-D-glucopyranoside. After 96 hours of reaction, the molar yield of 2-phenylethanol-β-D-glucopyranoside relative to 2-phenylethanol was 46.6%, and the reaction selectivity was 81.6%.

[0070] <Test Example 4: Substrate specificity of recombinant RpG3> The recombinant E. coli into which the RpG3 gene was introduced was inoculated into LB medium (50 mL) containing 50 μg / mL ampicillin, and the OD 600It was shake-cultured at 37 °C and 160 rpm for about 3 hours until the OD reached 0.6. Gene expression was induced by adding isopropylthiogalactoside (IPTG) at a final concentration of 0.1 mmol / L. Furthermore, the recombinant Escherichia coli was cultured at 18 °C for 1 day. 1.5 mL of the obtained culture solution was dispensed into a 2-mL microtube, centrifuged at 4 °C and 15,000 rpm for 5 minutes, and 0.75 mL of phosphate buffer (20 mM KPB, pH 7.0) was added to the obtained cells for washing. Centrifugation was performed again under the same conditions as above, and the supernatant was removed to prepare washed cells.

[0071] To the washed cells in the microtube, 0.3 mL of a phosphate buffer (20 mM KPB, pH 7.0) containing 1.0 g / L of a substrate compound (nerol, 1-butanol, 1-octanol) and 100 g / L of cellobiose was added as a reaction solution, and the reaction was carried out at 40 °C and 190 rpm for 24 hours. After adding 0.3 mL of methanol to stop the reaction, centrifugation was performed, and the supernatant was analyzed by thin-layer chromatography (TLC). As a result, the production of β-glucosides corresponding to each substrate compound could be confirmed.

[0072] <Test Example 5: Expression of Recombinant EaG3 and Glycosylation Reaction> The recombinant Escherichia coli into which the EaG3 gene was introduced was inoculated into an LB medium (50 mL) containing 50 μg / mL ampicillin, and the OD of the culture solution 600 It was shake-cultured at 37 °C and 160 rpm for about 3 hours until the OD reached 0.6. Gene expression was induced by adding isopropylthiogalactoside (IPTG) at a final concentration of 0.5 mmol / L. Furthermore, the recombinant Escherichia coli was cultured at 25 °C for 1 day. 1.5 mL of the obtained culture solution was dispensed into a 2-mL microtube, centrifuged at 4 °C and 15,000 rpm for 5 minutes, and 0.75 mL of phosphate buffer (20 mM KPB, pH 7.0) was added to the obtained cells for washing. Centrifugation was performed again under the same conditions as above, and the supernatant was removed to prepare washed cells.

[0073] To 0.3 mL of the washed cells in the microtube, 0.3 mL of a phosphate buffer (20 mM KPB, pH 7.0) containing 7.0 g / L of 2-phenylethanol and 100 g / L of cellobiose was added as a reaction solution, and the reaction was carried out at 40 °C and 190 rpm for 24 hours. After adding 0.3 mL of methanol to stop the reaction, centrifugation was performed, and the supernatant was analyzed by TLC and HPLC. As a result, the production of 2-phenylethanol-β-D-glucopyranoside could be confirmed.

[0074] <Test Example 6: Glycosylation Reaction of 2-Phenylethanol by Various Microorganisms> In Test Example 6, as microorganisms producing β-glycosidase, Aspergillus tubingensis JCM 5697 strain, Talaromyces albobiverticillius NBRC 6580 strain, Penicillium janthinellum NBRC 4651 strain, Penicillium adametzioi NBRC 7680 strain, Phaeosphaeria oryzae NBRC 33076 strain, Pyrenophora dictyoides NBRC 7370 strain, Marasmiellus mesosporus NBRC 105515 strain, Omphalotus guepiniformis NBRC 6992 strain, Agrocybe cylindracea NBRC 9075 strain, Torula dematia NBRC 6212 strain, Glyphoaria frondosa NBRC 7040 strain, Aureobasidium pullulans NBRC 4465 strain, Pichia farinosa NBRC 0193 strain, Sibirindonea saturnus NBRC 0992 strain, Sibirindonea saturnus NBRC 0941 strain, Papiliotrema aurea NBRC 0372 strain, Daedalea dickinsii NBRC 31163 strain, Daedalea cerealis NBRC 9286 strain, Lentinu la edodes NBRC 8340 strain, and Coprinopsis cinerea NBRC 30628 strain, a total of 20 types were used.

[0075] Among the above microorganisms, for fungi, 1% glucose, 0.3% K 2 HPO 4 、0.02% MgSO 4 ·7H 2It was cultured in 500 mL of a medium containing O, 1.5% peptone, 0.2% NaCl, and 0.1% yeast extract at 28 °C and 300 rpm for 24 hours. Then, after centrifugation at 8000 rpm for 20 minutes, the obtained cells were suspended in 0.85% NaCl solution and centrifuged again at 8000 rpm for 20 minutes. Thereafter, the cells were spread on a petri dish and dried with a fan, and then ground in a mortar to prepare dried cells.

[0076] Also, for yeast, it was cultured in 500 mL of a medium containing 5% glucose, 0.5% peptone, 0.2% KH 2 PO 4 、0.1% K 2 HPO 4 、0.02% MgSO 4 ·7H 2 O, and 0.1% yeast extract at 28 °C and 300 rpm for 24 hours. Then, after centrifugation at 8000 rpm for 20 minutes, the obtained cells were suspended in 0.85% NaCl solution and centrifuged again at 8000 rpm for 20 minutes. Thereafter, the cells were spread on a petri dish and dried with a fan, and then ground in a mortar to prepare dried cells.

[0077] Also, for basidiomycetes, it was cultured in 500 mL of a medium containing 0.4% yeast extract, 1.0% malt extract, and 0.4% glucose at 28 °C and 300 rpm for 24 hours. Then, after centrifugation at 8000 rpm for 20 minutes, the obtained cells were suspended in 0.85% NaCl solution and centrifuged again at 8000 rpm for 20 minutes. Thereafter, the cells were spread on a petri dish and dried with a fan, and then ground in a mortar to prepare dried cells.

[0078] To 0.5 mL of a phosphate buffer (20 mM KPB, pH 6.0) containing 7.0 g / L of 2-phenylethanol and 100 g / L of cellobiose, 0.5 mL was added as a reaction solution to the dried cells of each microorganism prepared above, and the mixture was incubated at 45 °C for 24 hours while shaking at 1200 strokes / min. The resulting reaction solution was centrifuged at 20 °C and 10,000 rpm for 1 minute, and the supernatant was recovered. Then, the supernatant was analyzed by HPLC, and the accumulation amount of the glycoside (2-phenylethanol-β-D-glucopyranoside) was quantified. Specifically, 2-phenylethanol-β-D-glucopyranoside whose structure was determined by NMR analysis was used as a standard, and calibration curves were prepared by analyzing standard solutions of various concentrations by HPLC. The accumulation amount of the glycoside was quantified from the area value of the peak showing the same retention time as the standard.

[0079] HPLC analysis was performed using an HPLC system (Shimadzu) equipped with a COSMOSIL 5C 18 -AR-II column (4.6 × 150 mm; Nacalai Tesque). For gradient elution, 5.7 × 10 -2 %(v / v) acetic acid (eluent A) and acetonitrile (eluent B) were used. The flow rate was 1.2 mL / min. The gradient conditions were as follows: 0 - 5 minutes: 10% eluent B, 5 - 16 minutes: linear gradient of eluent B from 0 to 80%, 16 - 17 minutes: linear gradient of eluent B from 80 to 100, 17 - 22 minutes: 100% eluent B, 22 - 23 minutes: linear gradient of eluent B from 100 to 10%, 23 - 27 minutes: 10% eluent B. The injector needle of the autosampler was washed with methanol to prevent coagulation of insoluble components after sample injection. For monitoring the eluate, an SPD0-M10A, M20A, or M40A photodiode array detector (Shimadzu) was used. The results are shown in Table 3 below.

[0080]

Table 3

Claims

1. A method for producing a β-glucosyl glycoside, comprising allowing any one of the microorganisms (1) to (24) below or a β-glycosidase derived from the microorganism to act on a system containing a substrate compound having a hydroxyl group and a glucose donor having a β-glycosidic bond, and recovering the β-glucosyl glycoside of the substrate compound. (1) Ensifer adhaerens (2) Shinella yambaruensis (3) Shinella granuli (4) Shinella zoogloeoides (5) Aspergillus luchuensis (6) Aspergillus tubingensis (7) Talaromyces albobiverticillius (8) Penicillium janthinellum (9) Penicillium adametzii (10) Phaeosphaeria oryzae (11) Pyrenophora dictyoides (12) Marasmiellus mesosporus (13) Omphalotus guepiniiformis (14) Agrocybe cylindracea (15) Torula dematia (16) Grifola frondosa (17) Aureobasidium pullulans (18) Pichia farinosa (19) Cyberlindnera saturnus (20) Papiliotrema aurea (21) Daedalea dickinsii (22) Daedalea serialis (23) Lentinula edodes (24) Coprinopsis cinerea

2. A transformant in which a gene encoding a β-glucosidase derived from any one of the following microorganisms (1) to (28) is introduced is allowed to act on a system containing a substrate compound having a hydroxyl group and a glucose donor having a β-glycosidic bond, and the β-glucoside of the substrate compound is recovered. A method for producing a β-glucosyl glycoside, which comprises: (1) Ensifer adhaerens (2) Shinella yambaruensis (3) Shinella granuli (4) Shinella zoogloeoides (5) Aspergillus luchuensis (6) Aspergillus tubingensis (7) Talaromyces albobiverticillius (8) Penicillium janthinellum (9) Penicillium adametzii (10) Phaeosphaeria oryzae (11) Pyrenophora dictyoides (12) Marasmiellus mesosporus (13) Omphalotus guepiniiformis (14) Agrocybe cylindracea (15) Torula dematia (16) Grifola frondosa (17) Aureobasidium pullulans (18) Pichia farinosa (19) Cyberlindnera saturnus (20)Papiliotrema aurea (21)Daedalea dickinsii (22)Daedalea serialis (23)Lentinula edodes (24)Coprinopsis cinerea (25)Rhizobium radiobacter (26)Rhizobium pusense (27)Rhizobium daejeonense (28)Rhizobium paknamense

3. A method for producing a β - glucoside, comprising introducing a gene encoding any of the following proteins (a) to (c) into a system containing a substrate compound having a hydroxyl group and a glucose donor having a β - glycosidic bond, or allowing any of the following proteins (a) to (c) to act, and recovering the β - glucoside of the substrate compound. (a) A protein consisting of the amino acid sequence of SEQ ID NO: 1 or 2. (b) A protein consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, or added in the amino acid sequence of (a) and having β - glucosidase activity. (c) A protein having a sequence identity of 90% or more with the amino acid sequence of (a) and having β - glucosidase activity.

4. The method for producing a β - glucoside according to any one of claims 1 to 3, wherein the glucose donor is cellobiose.

5. A transformant into which a gene encoding any of the following proteins (a) to (c) has been introduced. (a) A protein consisting of the amino acid sequence of SEQ ID NO: 1 or 2. (b) A protein consisting of an amino acid sequence in which one or several amino acid residues are substituted, deleted, or added in the amino acid sequence of (a) and having β - glucosidase activity. (c) A protein having a sequence identity of 90% or more with the amino acid sequence of (a) and having β - glucosidase activity.

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