General acid-base catalytic mutant enzyme of α-glucosidase

Abstract

To provide an enzyme that can catalyze transglycosylation reactions.SOLUTION: Provided is a protein that contains any of the following amino acid sequences, does not have hydrolytic activity against α-glucoside, and has the activity of catalyzing transglycosylation reactions using activated sugars as donor substrates: an amino acid sequence in which the amino acid residue at position 660 is substituted, or a mutant sequence thereof, in the amino acid sequence of α-glucosidase (ANG) derived from Aspergillus niger; an amino acid sequence in which an amino acid residue at a position corresponding to position 660 in the ANG amino acid sequence is substituted when the amino acid sequence is aligned with the ANG amino acid sequence, or a mutant sequence thereof, in the amino acid sequence of α-glucosidase that has 60% or more sequence identity with the amino acid sequence of ANG; and an amino acid sequences in which general acid-base catalytic residues are substituted, or a mutant sequence thereof, in the amino acid sequence of α-glucosidase that has 60% or more sequence identity with the amino acid sequence of ANG.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a general acid-base catalytic mutant enzyme of α-glucosidase and a method for producing a transglycosylation product using the same. [Background technology]

[0002] α-Glucosidase (EC 3.2.1.20) is an enzyme that uses α-glucosides such as maltooligosaccharides, dextrins, and sucrose as substrates and hydrolyzes the α-glucosidic bond at the non-reducing end to generate α-glucose. In addition, when an acceptor other than water is present, it also catalyzes a glycosyltransferase reaction to generate α-glucosides by adding an α-glucosyl group to the acceptor. Methods for synthesizing α-glucosides using the glycosyltransferase reaction of α-glucosidase have been reported (e.g., Patent Document 1, Patent Document 2, Patent Document 3). On the other hand, in the case of glycoside synthesis using an enzyme such as α-glucosidase that has both hydrolysis and transglycosylation activities, the glycoside produced by the transglycosylation reaction becomes a substrate for the enzyme and is hydrolyzed, so the transglycosylation reaction product may not accumulate in large amounts. To solve this problem, the glycosynthase reaction was developed (Non-Patent Document 1). The glycosynthase reaction is a reaction catalyzed by a mutant enzyme in which the nucleophilic catalytic residue of an anomer-retaining glycosyl hydrolase has been replaced with a non-nucleophilic amino acid such as glycine, alanine, or serine, and uses an activated sugar that is the opposite anomeric form to the original substrate as a glycosyl donor. In the presence of a suitable acceptor, a glycosyltransferase product is synthesized. Since the mutation has been introduced into the nucleophilic catalytic residue, hydrolysis activity is lost, and the glycosyltransferase product accumulates without being hydrolyzed. Many glycosyltransferase reactions involving glycosylhydrolases have been reported so far (Non-Patent Documents 2, 3, 4, etc.). It has been reported that even in reactions using enzymes with mutated nucleophilic catalytic residues, in the presence of nucleophiles such as azide ions and formate ions, these nucleophiles complement the function of the nucleophilic catalytic residues (chemical rescue reaction), making it possible to use the same anomeric activated sugar as the original substrate as a donor, thereby obtaining transglycosylation products in high yields. However, it has been reported that azide sugars and sugar formates accumulate in chemical rescue reactions (Non-Patent Documents 5, 6, 7, and 8). [Prior art document] [Patent documents]

[0003] [Patent Document 1] Patent No. 5992902 [Patent Document 2] Patent No. 4197604 [Patent Document 3] Patent No. 2832848 [Non-patent literature]

[0004] [Non-patent document 1] Mackenzie LF, Wang QP, Warren RAJ, Withers SG. Glycosynthases: Mutant glycosidases for oligosaccharide synthesis. J Am Chem Soc . 1998;120(22):5583-5584. doi: 10.1021 / ja980833d. [Non-Patent Document 2] Hayes MR, Pietruszka J. Synthesis of Glycosides by Glycosynthases. Molecules. 2017;22(3):1434. doi: 10.3390 / molecules22091434. [Non-patent document 3] Okuyama M, Mori H, Watanabe K, Kimura A, Chiba S. Alpha-glucosidase mutant catalyzes “alpha-glycosynthase”-type reaction. Biosci Biotechnol Biochem. 2002;66(4):928-33. doi: 10.1271 / bbb.66.928. [Unauthorized literature 4] Yamamoto K, Davis BG. Creation of an α-mannosynthase from a broad glycosidase scaffold. Angew Chem Int Ed Engl. 2012;51(30):7449-53. doi: 10.1002 / anie.201201081. [Unauthorized document 5] Moracci M, Trincone A, Perugino G, Ciaramella M, Rossi M. Restoration of the activity of active-site mutants of the hyperthermophilic beta-glycosidase from Sulfolobus solfataricus: dependence of the mechanism on the action of external nucleophiles. Biochemistry. 1998;37(49):17262-70. doi: 10.1021 / bi981855f. [Unauthorized literature 6] Okuyama M, Matsunaga K, Watanabe KI, Yamashita K, Tagami T, Kikuchi A, Ma M, Klahan P, Mori H, Yao M, Kimura A. Efficient synthesis of α-galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α-galactosidase (BtGH97b). FEBS J. 2017;284(5):766-783. doi: 10.1111 / febs.14018. [Non-patented document 7] Korn Minko, Research on the molecular structure of glycolytic enzymes derived from bacteria and its application, Ph.D. thesis, Hokkaido University (2009) [ Abstract ]Viladot JL, de Ramon E, Durany O, Planas A. Probing the mechanism of Bacillus 1,3-1,4-beta-D-glucan 4-glucanohydrolases by chemical rescue of inactive mutants at catalytically essential residues. Biochemistry. 1998;37(32):11332-42. doi: 10.1021 / bi980586q. [ PubMed ] Jahn M, Marles J, Warren RA, Withers SG. Thioglycoligases: mutant glycosidases for thioglycoside synthesis. Angew Chem Int Ed Engl. 2003;42(3):352-4. doi: 10.1002 / anie.200390114. [ PubMed ] 10]Kim YW, Lovering AL, Chen H, Kantner T, McIntosh LP, Strynadka NC, Withers SG. Expanding the thioglycoligase strategy to the synthesis of alpha-linked thioglycosides allows structural investigation of the parent enzyme / substrate complex. J Am Chem Soc. 2006;128(7):2202-3. doi: 10.1021 / ja057904a. [Unauthorized literature 11] Kim YW, Zhang R, Chen H, Withers SG. O-glycoligases, a new category of glycoside bond-forming mutant glycosidases, catalyse facile syntheses of isoprimeverosides. Chem Commun (Camb). 2010;46(46):8725-7. doi: 10.1039 / c0cc03168b. [Unauthorized literature 12] Li C, Ahn HJ, Kim JH, Kim YW. Transglycosylation of engineered cyclodextrin glucanotransferases as O-glycoligases. Carbohydr Polym. 2014;99:39-46. doi: 10.1016 / j.carbpol.2013.08.056. [Unauthorized literature 13] Chao Li, Jin-Hyo Kim, Young-Wan Kim. α-Thioglycoligase-based synthesis of O-aryl α-glycosides as chromogenic substrates for α-glycosidases. J Mol Catl B-Enzym. 2013;87:24-9. doi: 10.1016 / j.molcatb.2012.10.008. [Unlicensed Document 14] TIFF2024009555000001.tif32160 [Non-Patent Document 15] Okuyama M, Okuno A, Shimizu N, Mori H, Kimura A, Chiba S. Carboxyl group of residue Asp647 as possible proton donor in catalytic reaction of alpha-glucosidase from Schizosaccharomyces pombe. Eur J Biochem. 2001;268(8):2270-80. doi: 10.1046 / j.1432-1327.2001.02104.x. Summary of the Invention [Problem to be solved by the invention]

[0005] The glycoligase reaction has been developed as an efficient method for synthesizing transglycosylation products to replace the glycosynthase reaction (Non-Patent Document 9). The glycoligase reaction is a reaction catalyzed by a mutant enzyme in which the general acid-base catalytic residue of a glycosyl hydrolase is replaced with alanine, asparagine, glutamine, etc., and uses an activated sugar such as 1-fluoro-1-deoxy sugar of the same anomeric form as the original substrate as a sugar donor, and can synthesize a transglycosylation product in the presence of a suitable acceptor. Because a mutation has been introduced into the general acid-base catalytic residue, hydrolysis activity has been lost, and the transglycosylation product accumulates without being hydrolyzed. Jahn et al. reported the synthesis of β-thioglucosyl oligosaccharides and β-thiomannosyl oligosaccharides by the glycoligase reaction using a β-glucosidase enzyme with a general acid-base catalytic residue mutant and 2,4-dinitrophenyl β-glucoside as the sugar donor, and by the glycoligase reaction using a β-mannosidase enzyme with a general acid-base catalytic residue mutant and 2,5-dinitrophenyl β-mannoside as the sugar donor, respectively (Non-Patent Document 9). Kim et al. reported the synthesis of α-thioglucosyl oligosaccharides and α-thioxylosyl oligosaccharides by the glycoligase reaction using a mutant enzyme of the general acid-base catalytic residue of α-glucosidase derived from Sulfolobus solfataricus and 1-deoxy-1-fluoro-α-glucose as the sugar donor, and the glycoligase reaction using a mutant enzyme of the general acid-base catalytic residue of α-xylosidase and 1-deoxy-1-fluoro-α-xylose as the sugar donor, respectively (Non-Patent Document 10). All of these four synthetic reactions use highly nucleophilic thiosugars as the acceptor, and are specifically called thioglycoligase reactions among glycoligase reactions. Although there have been other reports on the glycoligase reaction (Non-Patent Documents 11, 12, 13, and 14), all of the glycosynthase reactions and glycoligase reactions known so far use activated sugars derived from the same sugar as the original substrate as the donor. An object of the present application is to provide an enzyme capable of catalyzing a glycosyltransferase reaction, preferably an enzyme capable of using, as a donor, an activated sugar derived from a sugar different from the original substrate. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems, and have surprisingly found that a mutant enzyme in which the general acid-base catalytic residue of α-glucosidase derived from Aspergillus niger has the activity of catalyzing the transglycosylation reaction using activated sugars derived from α-mannose and α-galactose, which are not the original substrates, as glycosyl donors, in addition to activated sugars derived from α-glucose, which is the original substrate, as glycosyl donors, thereby completing the present invention. Even more surprisingly, the present inventors have found that the transglycosylation reaction of the mutant enzyme can use a very wide range of compounds as acceptors. The aspects of the present invention are described in detail below.

[0007] [Section 1] A protein comprising any one of the following amino acid sequences (a) to (g), which does not have hydrolytic activity against α-glucosides and has activity to catalyze a glycosyltransferase reaction using an activated sugar as a donor substrate: (a) an amino acid sequence in which the amino acid residue at position 660 in the amino acid sequence of SEQ ID NO:2 has been substituted; (b) an amino acid sequence in which the amino acid residue at position 660 in the amino acid sequence of SEQ ID NO:2 has been substituted and further in which one or more amino acid residues have been substituted, deleted, inserted and / or added; (c) an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, wherein the amino acid residue at the position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is an amino acid residue other than an aspartic acid residue; (d) an amino acid sequence of an α-glucosidase having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO: 2, in which an amino acid residue at a position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is substituted; (e) an amino acid sequence of an α-glucosidase having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, in which an amino acid residue at a position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is substituted and further in which one or more amino acid residues are substituted, deleted, inserted and / or added; (f) an amino acid sequence of an α-glucosidase having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO:2, in which a general acid-base catalytic residue has been substituted. (g) An amino acid sequence of an α-glucosidase having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO:2, in which a general acid-base catalytic residue has been replaced and one or more further amino acid residues have been replaced, deleted, inserted and / or added. [Section 2] A protein comprising any one of the following amino acid sequences (a) to (g), which does not have hydrolytic activity against α-glucosides and has activity to catalyze a glycosyltransferase reaction using an activated sugar as a donor substrate: (a) an amino acid sequence in which the amino acid residue at position 660 in the amino acid sequence of SEQ ID NO:2 is substituted with an amino acid residue selected from the group consisting of an asparagine residue, an alanine residue, a serine residue, a glycine residue, and a glutamine residue; (b) an amino acid sequence in which the amino acid residue at position 660 in the amino acid sequence of SEQ ID NO:2 is substituted with an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues, and further in which one or more amino acid residues are substituted, deleted, inserted, and / or added; (c) an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO:2, wherein the amino acid residue at the position corresponding to position 660 in the amino acid sequence of SEQ ID NO:2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO:2 is an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues; (d) an amino acid sequence of an α-glucosidase having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO: 2, in which an amino acid residue at a position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is substituted with an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues; (e) an amino acid sequence of an α-glucosidase having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO: 2, in which an amino acid residue at a position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is substituted with an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues, and further in which one or more amino acid residues have been substituted, deleted, inserted, and / or added; (f) An amino acid sequence of an α-glucosidase having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO:2, in which a general acid-base catalytic residue is replaced with an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues. (g) an amino acid sequence of an α-glucosidase having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO: 2, in which a general acid-base catalytic residue is replaced with an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues, and in which one or more amino acid residues are further substituted, deleted, inserted, and / or added. The protein according to [Item 1], [Section 3] A protein according to [Item 1] or [Item 2], comprising the amino acid sequence of SEQ ID NO:3. [Section 4] A protein comprising any one of the following amino acid sequences (a) to (c), which does not have hydrolytic activity against α-glucosides and has activity to catalyze a glycosyltransferase reaction using an activated sugar as a donor substrate: (a) the amino acid sequence of SEQ ID NO:3 (b) an amino acid sequence in which one or more amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, and in which the amino acid residue at the position corresponding to position 635 in the amino acid sequence of SEQ ID NO: 3 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 3 is an amino acid residue other than an aspartic acid residue; (c) an amino acid sequence having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at the position corresponding to position 635 in the amino acid sequence of SEQ ID NO: 3 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 3 is an amino acid residue other than an aspartic acid residue. [Section 5] A protein comprising any one of the following amino acid sequences (a) to (c), which does not have hydrolytic activity against α-glucosides and has activity to catalyze a glycosyltransferase reaction using an activated sugar as a donor substrate: (a) the amino acid sequence of SEQ ID NO:3 (b) an amino acid sequence in which one or more amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, and in which the amino acid residue at the position corresponding to position 635 in the amino acid sequence of SEQ ID NO: 3 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 3 is an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues and glutamine residues; (c) an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at the position corresponding to position 635 in the amino acid sequence of SEQ ID NO: 3 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 3 is an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues. The protein according to [Item 4], [Section 6] The protein according to any one of [Item 1] to [Item 5], wherein the activated sugar is derived from a sugar selected from the group consisting of α-glucose, α-mannose, and α-galactose. [Section 7] The activated sugar is 1-fluoro-1-deoxy-α-glucose, 1-bromo-1-deoxy-α-glucose, 1-chloro-1-deoxy-α-glucose, 4-nitrophenyl α-glucoside, 2,4-dinitrophenyl α-glucoside, α-glucose-1-phosphate, α-uridine diphosphate glucoside, 4-Methylumbelliferyl α-glucoside, 1-fluoro-1-deoxy-α-mannose, 1-bromo-1-deoxy-α-mannose, 1-chloro-1-deoxy-α-mannose, 4-nitrophenyl α-mannoside, 2,4-dinitrophenyl α-mannoside, α-mannose-1-phosphate, α-uridine diphosphate mannoside, 4-Methylumbelliferyl α-mannoside, 1-Fluoro-1-deoxy-α-galactose 1-bromo-1-deoxy-α-galactose, 1-chloro-1-deoxy-α-galactose, 4-nitrophenyl α-galactoside, 2,4-dinitrophenyl α-galactoside, α-galactose-1-phosphate, α-uridine diphosphate galactoside, and 4-Methylumbelliferyl α-galactoside The protein according to any one of [Item 1] to [Item 6], which is selected from the group consisting of: [Section 8] The protein according to any one of [Item 1] to [Item 7], wherein the activated sugar is derived from a sugar selected from the group consisting of α-mannose and α-galactose. [Section 9] The protein according to any one of [Item 1] to [Item 3] and [Item 6] to [Item 8], wherein the α-glucosidase is an α-glucosidase classified into Glycoside Hydrolase Family 31. [Section 10] The protein according to any one of [Item 1] to [Item 3] and [Item 6] to [Item 9], wherein the α-glucosidase is derived from the genus Aspergillus, Sulfolobus, Schwanniomyces, Saccharomyces, Schizosaccharomyces, Bacteroides, Acremonium, or Podospora. [Section 11] The protein according to any one of [Item 1] to [Item 10], which consists of the amino acid sequence of SEQ ID NO:1. [Section 12] The protein according to any one of [Item 1] to [Item 11], wherein an acceptor substrate in the glycosyltransfer reaction is a compound selected from the group consisting of a carboxylic acid compound, a phosphate compound, and a compound having a thiol group. [Section 13] An enzyme preparation comprising the protein according to any one of [Item 1] to [Item 12]. [Section 14] The enzyme preparation according to [Item 13], for catalyzing a glycosyltransfer reaction using, as a donor substrate, an activated sugar derived from a sugar selected from the group consisting of α-mannose and α-galactose. [Section 15] A polynucleotide encoding the protein according to any one of [1] to

[12] . [Section 16] A vector comprising the polynucleotide according to [Item 15]. [Section 17] A host cell transformed with the vector described in [Item 16]. [Section 18] A method for producing the protein according to any one of [Item 1] to [Item 12], comprising culturing the host cell according to [Item 17] and recovering the protein according to any one of [Item 1] to [Item 12] from the culture. [Section 19] A method for transferring sugar from a donor substrate to an acceptor substrate, the method comprising the step of reacting a donor substrate, which is an activated sugar, with an acceptor substrate in the presence of the protein according to any one of [Item 1] to [Item 12] or the enzyme agent according to [Item 13] or [Item 14]. [Section 20] A method for producing a transglycosylation product, comprising a step of reacting a donor substrate, which is an activated sugar, with an acceptor substrate in the presence of the protein according to any one of [Item 1] to [Item 12] or the enzyme preparation according to [Item 13] or [Item 14]. [Section 21] The method according to [Item 19] or [Item 20], wherein the activated sugar is derived from a sugar selected from the group consisting of α-glucose, α-mannose, and α-galactose. [Section 22] The activated sugar is 1-fluoro-1-deoxy-α-glucose, 1-bromo-1-deoxy-α-glucose, 1-chloro-1-deoxy-α-glucose, 4-nitrophenyl α-glucoside, 2,4-dinitrophenyl α-glucoside, α-glucose-1-phosphate, α-uridine diphosphate glucoside, 4-Methylumbelliferyl α-glucoside, 1-fluoro-1-deoxy-α-mannose, 1-bromo-1-deoxy-α-mannose, 1-chloro-1-deoxy-α-mannose, 4-nitrophenyl α-mannoside, 2,4-dinitrophenyl α-mannoside, α-mannose-1-phosphate, α-uridine diphosphate mannoside, 4-Methylumbelliferyl α-mannoside, 1-Fluoro-1-deoxy-α-galactose 1-bromo-1-deoxy-α-galactose, 1-chloro-1-deoxy-α-galactose, 4-nitrophenyl α-galactoside, 2,4-dinitrophenyl α-galactoside, α-galactose-1-phosphate, α-uridine diphosphate galactoside, and 4-Methylumbelliferyl α-galactoside The method according to any one of [Item 19] to [Item 21], wherein the method is selected from the group consisting of: [Section 23] The method according to any one of [Item 19] to [Item 22], wherein the activated sugar is derived from a sugar selected from the group consisting of α-mannose and α-galactose. [Section 24] The method according to any one of [Item 19] to [Item 23], wherein the receptor substrate is a compound selected from the group consisting of a carboxylic acid compound, a phosphate compound, and a compound having a thiol group. [Section 25] A method for producing a food, feed, cosmetic, reagent, or pharmaceutical, comprising the steps of obtaining a transglycosylation product by the method according to any one of [Item 20] to [Item 24], and obtaining a food, feed, cosmetic, or pharmaceutical from the transglycosylation product obtained in the above step. Effect of the Invention

[0008] According to the present invention, it is possible to provide a protein that does not have hydrolytic activity against α-glucosides and has activity to catalyze a glycosyltransfer reaction using an activated sugar as a donor substrate. [Brief description of the drawings]

[0009] [Figure 1]Figure 1 shows the thin-layer chromatograms of the reaction with fatty acids. Standard: mannose, 1-fluoro-1-deoxy-α-mannose -: negative control, +: enzyme reaction. The star indicates that the transglycosylation product was detected. [Figure 2-1] Figure 2-1 shows the structural formula and molecular weight of 1-O-mannose formate. [Figure 2-2] FIG. 2-2 shows the results of ESI-MS analysis of the reaction product with formic acid. [Figure 3-1] Figure 3-1 shows the structural formula and molecular weight of 1-O-acetylmannose. [Figure 3-2] FIG. 3-2 shows the results of ESI-MS analysis of the reaction products with acetic acid. [Figure 4-1] Figure 4-1 shows the structural formula and molecular weight of 1-O-mannose butyrate. [Figure 4-2] FIG. 4-2 shows the results of ESI-MS analysis of the reaction product with butyric acid. [Diagram 5] Figure 5 shows thin-layer chromatograms of the reaction with acetic acid at different pH values. Standards: mannose, 1-fluoro-1-deoxy-α-mannose → indicates the product presumed to be 1-fluoro-1-deoxy-α-mannose, and the star indicates 1-O-acetylmannose. [Figure 6-1] Figure 6-1 shows the structural formula and molecular weight of mannosyl octanoic acid. [Figure 6-2] FIG. 6-2 shows the results of ESI-MS analysis of the reaction product with octanoic acid. [Figure 7] Figure 7 shows the thin-layer chromatograms of the reaction with plant hormones. Standard: mannose, 1-fluoro-1-deoxy-α-mannose -: negative control, +: enzyme reaction. The star indicates that the transglycosylation product was detected. [Figure 8-1] Figure 8-1 shows the structural formula and molecular weight of mannosyl ascorbic acid. [Figure 8-2] FIG. 8-2 shows the results of ESI-MS analysis of the reaction product with ascorbic acid. [Figure 9-1] Figure 9-1 shows the structural formula and molecular weight of mannosylindole acetic acid. [Figure 9-2]FIG. 9-2 shows the results of ESI-MS analysis of the reaction product with indole acetic acid. [Figure 10-1] Figure 10-1 shows the structural formula and molecular weight of mannosyl jasmonic acid. [Figure 10-2] FIG. 10-2 shows the results of ESI-MS analysis of the reaction products with jasmonic acid. [Figure 11-1] Figure 11-1 shows the structural formula and molecular weight of mannosyl abscisic acid. [Figure 11-2] FIG. 11-2 shows the results of ESI-MS analysis of the reaction products with abscisic acid. [Figure 12] Figure 12 shows the thin-layer chromatograms in the reaction with salicylic acid (pH 4.0). Standard: mannose, 1-fluoro-1-deoxy-α-mannose. -: negative control, +: enzyme reaction. The star indicates that a glycosyltransferase reaction product was detected. [Figure 13-1] Figure 13-1 shows the structural formula and molecular weight of mannosyl salicylic acid. [Figure 13-2] FIG. 13-2 shows the results of ESI-MS analysis of the reaction product with salicylic acid. [Figure 14] Figure 14 shows the thin-layer chromatograms of the reaction with organic acids. -: negative control, +: enzyme reaction reaction time was 6 hours. However, the reaction time for pyrophosphate alone was 96 hours. The star indicates that a glycosyltransferase product was detected. [Figure 15] Figure 15 shows the thin-layer chromatograms of the time course of the reaction with organic acids. Standard: mannose, 1-fluoro-1-deoxy-α-mannose -: negative control, +: enzyme reaction. The star indicates that a glycosyltransferase reaction product was detected. [Figure 16-1] Figure 16-1 shows the structural formula and molecular weight of mannosylmalonic acid. [Figure 16-2] FIG. 16-2 shows the results of ESI-MS analysis of the reaction product with malonic acid. [Figure 17-1] Figure 17-1 shows the structural formula and molecular weight of mannosyl lactic acid. [Figure 17-2] FIG. 17-2 shows the results of ESI-MS analysis of the reaction product with L-lactic acid. [Figure 18-1] Figure 18-1 shows the structural formula and molecular weight of mannosylglyceric acid. [Figure 18-2] FIG. 18-2 shows the results of ESI-MS analysis of the reaction product with glyceric acid. [Figure 19-1] Figure 19-1 shows the structural formula and molecular weight of mannosyl pyrophosphate. [Figure 19-2] FIG. 19-2 shows the results of ESI-MS analysis of the reaction product with pyrophosphate. [Figure 20-1] Figure 20-1 shows the structural formula and molecular weight of mannosyl phthalic acid. [Figure 20-2] FIG. 20-2 shows the results of ESI-MS analysis of the reaction products with phthalic acid. [Figure 21-1] Figure 21-1 shows the structural formula and molecular weight of mannosyl citric acid. [Figure 21-2] FIG. 21-2 shows the results of ESI-MS analysis of the reaction products with citric acid. [Figure 22-1] Figure 22-1 shows the structural formula and molecular weight of mannosyl tartaric acid. [Figure 22-2] FIG. 22-2 shows the results of ESI-MS analysis of the reaction product with tartaric acid. [Figure 23] Figure 23 shows the thin-layer chromatograms of the reaction with amino acids. -: negative control, +: enzyme reaction. The reaction with CysMe was performed for 96 hours. The star indicates that the transglycosylation product was detected. [Figure 24-1] Figure 24-1 shows the structural formula and molecular weight of mannosylserine. [Figure 24-2] FIG. 24-2 shows the results of ESI-MS analysis of the reaction product with serine. [Figure 25-1] Figure 25-1 shows the structural formula and molecular weight of mannosylthreonine. [Figure 25-2] FIG. 25-2 shows the results of ESI-MS analysis of the reaction product with threonine. [Figure 26-1] Figure 26-1 shows the structural formula and molecular weight of mannosylcysteine. [Figure 26-2] FIG. 26-2 shows the results of ESI-MS analysis of the reaction product with cysteine. [Figure 27-1] Figure 27-1 shows the sequence and molecular weight of mannosyl-angiotensin I. "Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu" is SEQ ID NO: 10. [Figure 27-2] FIG. 27-2 shows the results of LC-MS analysis of the reaction products with angiotensin I. [Figure 28] Figure 28 shows the thin-layer chromatograms of the reaction with glucose derivative carboxylic acid. -: negative control, +: enzyme reaction. Reaction time was 48 hours. The star indicates that the transglycosylation product was detected. [Figure 29] Figure 29 shows a thin-layer chromatogram of the reaction using 1-fluoro-1-deoxy-α-glucose as a sugar donor. Standards: glucose, 1-fluoro-1-deoxy-α-glucose [Diagram 30] Figure 30 shows a thin-layer chromatogram of the reaction using 1-fluoro-1-deoxy-α-galactose as a glycosyl donor. Standard: Galactose, 1-fluoro-1-deoxy-α-galactose [Diagram 31] FIG. 31 shows the structural formulas of the glycosyl donors tested. [Diagram 32] Figure 32 shows the structural formulae of the tested fatty acids and the solvents used. Stars indicate those in which transglycosylation products were detected. [Diagram 33] Figure 32 shows the structural formulae of the plant hormones tested and the solvents used. Stars indicate those in which transglycosylation products were detected. [Diagram 34] Figure 34 shows the structural formulas of the organic acids tested. Stars indicate those in which transglycosylation products were detected. [Diagram 35] Figure 35 shows the structural formulas of the tested amino acids. Stars indicate those for which transglycosylation products were detected. [Diagram 36] Figure 36 shows the structural formulas of the glucose-derived carboxylic acids tested. Stars indicate those in which transglycosylation products were detected. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] In one aspect, the present application provides a method for producing a method for treating a cancer cell comprising: The present invention provides a protein (hereinafter sometimes referred to as the "protein of the present invention") that contains any one of the following amino acid sequences (a) to (g), has no hydrolytic activity against α-glucosides, and has activity to catalyze a transglycosylation reaction using an activated sugar as a donor substrate: (a) an amino acid sequence in which the amino acid residue at position 660 in the amino acid sequence of SEQ ID NO:2 has been substituted; (b) an amino acid sequence in which the amino acid residue at position 660 in the amino acid sequence of SEQ ID NO:2 has been substituted and further in which one or more amino acid residues have been substituted, deleted, inserted and / or added; (c) an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, wherein the amino acid residue at the position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is an amino acid residue other than an aspartic acid residue; (d) an amino acid sequence of an α-glucosidase having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO: 2, in which an amino acid residue at a position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is substituted; (e) an amino acid sequence of an α-glucosidase having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, in which an amino acid residue at a position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is substituted and further in which one or more amino acid residues are substituted, deleted, inserted and / or added; (f) an amino acid sequence of an α-glucosidase having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO:2, in which a general acid-base catalytic residue is substituted. (g) An amino acid sequence of an α-glucosidase having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO:2, in which a general acid-base catalytic residue has been replaced and further in which one or more amino acid residues have been replaced, deleted, inserted and / or added.

[0011] In one aspect, the present application provides a method for producing a method for treating a cancer cell comprising: The present invention provides a protein comprising any one of the following amino acid sequences (a) to (c), which does not have hydrolytic activity against α-glucosides and has activity to catalyze a transglycosylation reaction using an activated sugar as a donor substrate (hereinafter, the protein of this embodiment may also be referred to as the "protein of the present invention"): (a) the amino acid sequence of SEQ ID NO:3 (b) an amino acid sequence in which one or more amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, and in which the amino acid residue at the position corresponding to position 635 in the amino acid sequence of SEQ ID NO: 3 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 3 is an amino acid residue other than an aspartic acid residue; (c) an amino acid sequence having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at the position corresponding to position 635 in the amino acid sequence of SEQ ID NO: 3 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 3 is an amino acid residue other than an aspartic acid residue.

[0012] In one aspect, the present application provides an enzyme preparation containing the protein of the present invention (hereinafter, may be referred to as the "enzyme preparation of the present invention"). In the enzyme preparation of the present invention, the protein of the present invention may be contained in a microorganism expressing the protein of the present invention, or in the form of a culture of the microorganism.

[0013] In the present disclosure, the term "enzyme agent" refers to an agent used to catalyze a glycosyltransferase reaction in which an activated sugar is a donor substrate. The enzyme agent may be a protein itself, a microorganism expressing a protein itself, or a culture of a microorganism itself, or may contain other components (e.g., buffer solutions, pH adjusters; Triton X-100 (surfactant), DTT, TCEP (reducing agents), protease inhibitors, preservatives, glycerol (freezing agent), etc.) in addition to these.

[0014] In one aspect, the present application provides a method for transferring a sugar from a donor substrate, which is an activated sugar, to an acceptor substrate, comprising the step of reacting the donor substrate, which is an activated sugar, with the acceptor substrate in the presence of the protein of the present invention or the enzyme preparation of the present invention.

[0015] In one aspect, the present application provides a method for producing a transglycosylation product, comprising the step of reacting a donor substrate, which is an activated sugar, with an acceptor substrate in the presence of the protein of the present invention or the enzyme preparation of the present invention.

[0016] In one embodiment, the protein of the present invention comprises (or has) an amino acid sequence in which the amino acid residue at position 660 in the amino acid sequence of SEQ ID NO:2 is substituted, has no hydrolytic activity against α-glucosides, and has the activity of catalyzing a glycosyltransfer reaction using an activated sugar as a donor substrate. In one embodiment, the amino acid residue at position 660 is substituted with an amino acid residue selected from the group consisting of an asparagine residue, an alanine residue, a serine residue, a glycine residue, and a glutamine residue (e.g., an asparagine residue, an alanine residue, and a serine residue, e.g., an asparagine residue).

[0017] SEQ ID NO: 2 is the amino acid sequence of α-glucosidase derived from Aspergillus niger (UniProtID: A0PCH8). This α-glucosidase has an aspartic acid residue at position 660, which is a general acid-base catalytic residue. It is known that aspartic acid is completely conserved in closely related enzymes having a sequence identity of 60% or more with Aspergillus niger-derived α-glucosidase (UniProtID: A0PCH8) (SEQ ID NO: 2). [Table 1]

[0018] In one embodiment, the protein of the present invention comprises (or has) an amino acid sequence in which the amino acid residue at position 660 in the amino acid sequence of SEQ ID NO:2 has been substituted and further comprises substitution, deletion, insertion and / or addition of one or more amino acid residues, has no hydrolytic activity against α-glucosides, and has the activity of catalyzing a glycosyltransfer reaction using an activated sugar as a donor substrate. In one embodiment, the amino acid residue at position 660 is substituted with an amino acid residue selected from the group consisting of an asparagine residue, an alanine residue, a serine residue, a glycine residue, and a glutamine residue (e.g., an asparagine residue, an alanine residue, and a serine residue, e.g., an asparagine residue).

[0019] In the present disclosure, "the amino acid residue at position 660 is substituted and one or more additional amino acid residues are substituted, deleted, inserted and / or added" means that the amino acid residue at position 660 is substituted and one or more additional amino acid residues are substituted, deleted, inserted and / or added at a position other than the amino acid residue at position 660.

[0020] In one embodiment, the protein of the present invention comprises (or has) an amino acid sequence having a sequence identity of 60% or more (preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 96% or more, and even more preferably 97% or more) with the amino acid sequence of SEQ ID NO: 2, in which the amino acid residue at the position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is an amino acid residue other than an aspartic acid residue, and has no hydrolytic activity against α-glucosides and has the activity of catalyzing a glycosyltransfer reaction using an activated sugar as a donor substrate. In one embodiment, the amino acid residue at the position corresponding to position 660 is an amino acid residue selected from the group consisting of an asparagine residue, an alanine residue, a serine residue, a glycine residue, and a glutamine residue (e.g., an asparagine residue, an alanine residue, and a serine residue, e.g., an asparagine residue).

[0021] In one embodiment, the protein of the present invention comprises (or has) an amino acid sequence of an α-glucosidase having a sequence identity of 60% or more (preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more) to the amino acid sequence of SEQ ID NO: 2, in which the amino acid residue at the position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is substituted, and has no hydrolytic activity against α-glucosides and has the activity of catalyzing a glycosyltransfer reaction using an activated sugar as a donor substrate. In one embodiment, the amino acid residue at the position corresponding to position 660 is substituted with an amino acid residue selected from the group consisting of an asparagine residue, an alanine residue, a serine residue, a glycine residue, and a glutamine residue (e.g., an asparagine residue, an alanine residue, and a serine residue, e.g., an asparagine residue).

[0022] In one embodiment, the protein of the present invention comprises (or has) an amino acid sequence of an α-glucosidase that has a sequence identity of 60% or more (preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more) to the amino acid sequence of SEQ ID NO: 2, in which an amino acid residue at a position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is substituted, and further, one or more amino acid residues are substituted, deleted, inserted and / or added, and has no hydrolytic activity against α-glucosides and has the activity of catalyzing a glycosyltransfer reaction using an activated sugar as a donor substrate. In one embodiment, the amino acid residue at the position corresponding to position 660 is substituted with an amino acid residue selected from the group consisting of an asparagine residue, an alanine residue, a serine residue, a glycine residue, and a glutamine residue (e.g., an asparagine residue, an alanine residue, and a serine residue, e.g., an asparagine residue).

[0023] In the present disclosure, "the amino acid residue at the position corresponding to 660 in the amino acid sequence of SEQ ID NO: 2 has been substituted, and one or more additional amino acid residues have been substituted, deleted, inserted and / or added" means that the amino acid residue at the position corresponding to 660 has been substituted, and one or more additional amino acid residues have been substituted, deleted, inserted and / or added at positions other than the amino acid residue at 660.

[0024] In the present disclosure, "the amino acid residue at a position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when aligned with the amino acid sequence of SEQ ID NO: 2" means the amino acid residue at a position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when alignment is performed based on SEQ ID NO: 2 using a conventional method in the technical field (e.g., using a program such as Clustal Omega, T-coffee, MUSCLE, or PROMALS). Because the amino acid residues in the active center are subject to strong functional constraints and are conserved, when the amino acid sequence of SEQ ID NO: 2 is aligned with the amino acid sequence of another α-glucosidase, the amino acid residue at the position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 in that α-glucosidase is highly likely to be a general acid-base catalytic residue.

[0025] In one embodiment, the protein of the present invention comprises (or has) an amino acid sequence in which a general acid-base catalytic residue is substituted in the amino acid sequence of an α-glucosidase having a sequence identity of 60% or more (preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more) to the amino acid sequence of SEQ ID NO: 2, and has no hydrolytic activity against α-glucosides and has the activity of catalyzing a glycosyltransfer reaction using an activated sugar as a donor substrate. In one embodiment, the general acid-base catalytic residue is substituted with an amino acid residue selected from the group consisting of an asparagine residue, an alanine residue, a serine residue, a glycine residue, and a glutamine residue (e.g., an asparagine residue, an alanine residue, and a serine residue, e.g., an asparagine residue).

[0026] In one embodiment, the protein of the present invention comprises (or has) an amino acid sequence of an α-glucosidase having a sequence identity of 60% or more (preferably 70% or more, more preferably 80% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more) to the amino acid sequence of SEQ ID NO: 2, in which a general acid-base catalytic residue has been substituted and one or more amino acid residues have been further substituted, deleted, inserted and / or added, and the protein does not have hydrolytic activity against α-glucosides and has the activity of catalyzing a glycosyltransfer reaction using an activated sugar as a donor substrate. In one embodiment, the general acid-base catalytic residue is substituted with an amino acid residue selected from the group consisting of an asparagine residue, an alanine residue, a serine residue, a glycine residue, and a glutamine residue (e.g., an asparagine residue, an alanine residue, and a serine residue, e.g., an asparagine residue).

[0027] In the present disclosure, "a general acid-base catalytic residue is substituted and one or more amino acid residues are further substituted, deleted, inserted and / or added" means that the general acid-base catalytic residue is substituted and one or more amino acid residues are substituted, deleted, inserted and / or added at a position other than the general acid-base catalytic residue.

[0028] The general acid-base catalytic residues of α-glucosidase can be identified by conventional methods in the art, for example, according to the method described in Non-Patent Document 15.

[0029] In one embodiment, the protein of the present invention comprises (or has) the amino acid sequence of SEQ ID NO: 3, has no hydrolytic activity against α-glucosides, and has the activity of catalyzing a glycosyltransferase reaction using an activated sugar as a donor substrate. [Table 2]

[0030] In one embodiment, the protein of the present invention comprises (or has) an amino acid sequence in which one or more amino acid residues have been substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, and when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 3, the amino acid residue at the position corresponding to position 635 in the amino acid sequence of SEQ ID NO: 3 is an amino acid residue other than an aspartic acid residue, and has no hydrolytic activity against α-glucosides and has the activity of catalyzing a glycosyltransfer reaction using an activated sugar as a donor substrate. In one embodiment, the amino acid residue at the position corresponding to position 635 is an amino acid residue selected from the group consisting of an asparagine residue, an alanine residue, a serine residue, a glycine residue, and a glutamine residue (e.g., an asparagine residue, an alanine residue, and a serine residue, e.g., an asparagine residue).

[0031] In one embodiment, the protein of the present invention comprises (or has) an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 3, in which the amino acid residue at the position corresponding to position 635 in the amino acid sequence of SEQ ID NO: 3 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 3 is an amino acid residue other than an aspartic acid residue, and has no hydrolytic activity against α-glucosides and has the activity of catalyzing a glycosyltransfer reaction using an activated sugar as a donor substrate. In one embodiment, the amino acid residue at the position corresponding to position 635 is an amino acid residue selected from the group consisting of an asparagine residue, an alanine residue, a serine residue, a glycine residue, and a glutamine residue (e.g., an asparagine residue, an alanine residue, and a serine residue, e.g., an asparagine residue).

[0032] In the present specification, the type, number and position of the amino acid residues to be substituted, deleted, inserted and / or added are not particularly limited, so long as the protein of the present invention does not have hydrolytic activity against α-glucosides and has the activity of catalyzing a transglycosylation reaction using an activated sugar as a donor substrate. Examples of amino acid substitutions include conservative amino acid substitutions, but non-conservative amino acid substitutions may also be used, so long as the protein of the present invention does not have hydrolytic activity against α-glucosides and has the activity of catalyzing a transglycosylation reaction using an activated sugar as a donor substrate.

[0033] In the present disclosure, the range of "one to several" in "an amino acid sequence in which one or several amino acid residues are substituted, deleted, inserted and / or added" is not particularly limited, and may be, for example, 1 to 400, 1 to 300, 1 to 200, 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 10, 1 to 7, 1 to 5, or 1 to 3.

[0034] In this disclosure, amino acids are usually natural amino acids and are in the L-form, but can also include unnatural amino acids such as β-alanine, D-form amino acids, and modified amino acids modified by techniques such as alkylation, esterification, halogenation, etc.

[0035] In the present disclosure, the sequence identity of amino acid sequences may be a value expressed as a percentage of the frequency at which identical amino acid residues appear at the same site in amino acid sequences including gaps when two amino acid sequences to be compared are aligned and gaps are introduced as necessary to maximize the degree of amino acid identity between the two. The sequence identity of amino acids can be determined by using available computer software such as, for example, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. As a preferred example, the sequence identity of amino acid sequences can be determined by Protein BLAST (BLAST (登録商標)The results can be evaluated using the National Library of Medicine (NLM) program with default parameters.

[0036] An example of an "α-glucosidase having 60% or more sequence identity with the amino acid sequence of SEQ ID NO:2" is an α-glucosidase classified into Glycoside Hydrolase Family 31 that has 60% or more sequence identity with the amino acid sequence of SEQ ID NO:2. All α-glucosidases classified into Glycoside Hydrolase Family 31 have a general acid-base catalytic residue, Asp, at a position corresponding to position 660 in the amino acid sequence of SEQ ID NO:2.

[0037] In one embodiment, the "α-glucosidase having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2" is derived from the genus Aspergillus, Sulfolobus, Schwanniomyces, Saccharomyces, Schizosaccharomyces, Bacteroides, Acremonium, or Podospora. Preferably, it is derived from the genus Aspergillus (e.g., Aspergillus niger).

[0038] In one embodiment, the protein of the present invention has the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of the recombinant enzyme (ANG_D660N) obtained as described in the Examples of the present application, and includes an amino acid sequence (i.e., SEQ ID NO: 3) in which the general acid-base catalytic residue Asp660 of α-glucosidase derived from Aspergillus niger (UniProtID: A0PCH8) (SEQ ID NO: 2) has been replaced with asparagine and 25 amino acid residues at the N-terminus have been deleted, and an amino acid sequence (SEQ ID NO: 4: 23 amino acids) derived from the plasmid vector used in the preparation is added to the C-terminus of the amino acid sequence of SEQ ID NO: 3. The deleted N-terminal 25 amino acid residues are a predicted protein export signal sequence in Aspergillus niger. The additional sequence on the C-terminus (SEQ ID NO: 4, 23 amino acids) is a sequence called a myc epitope and a his tag sequence, which are amino acid sequences for specific detection with an anti-myc antibody and purification by metal affinity chromatography, respectively. As described in the Examples of this application, the inventors confirmed that the mutant enzyme (ANG_D660N) has the activity of catalyzing the transglycosylation reaction using an activated sugar as a donor substrate. Because the mutant enzyme (ANG_D660N) has a mutation in a general acid-base catalytic residue, the transglycosylation reaction product can accumulate without being hydrolyzed. [Table 3] [Table 4]

[0039] In one embodiment, the protein of the present invention comprises (or has) an amino acid sequence in which amino acid residues at positions corresponding to the N-terminal 25 amino acid residues in the amino acid sequence of SEQ ID NO:2 are deleted.

[0040] In the present disclosure, a "protein having an amino acid sequence" means that the primary structure of the protein is composed of the amino acid sequence. A "protein having an amino acid sequence" can mean a "protein consisting of the amino acid sequence."

[0041] In the present disclosure, a "protein containing an amino acid sequence" means that one or more amino acids may be added to the N-terminus and / or C-terminus of the amino acid sequence. The property of the protein of the present invention that "has no hydrolytic activity for α-glucosides and has activity to catalyze a transglycosylation reaction using an activated sugar as a donor substrate" may be due to the amino acid sequence portion, so that an amino acid sequence of one or more amino acids may be added to the N-terminus and / or C-terminus of the amino acid sequence, so long as the protein has no hydrolytic activity for α-glucosides and has activity to catalyze a transglycosylation reaction using an activated sugar as a donor substrate. For example, as long as the protein has no hydrolytic activity for α-glucosides and has activity to catalyze a transglycosylation reaction using an activated sugar as a donor substrate, an amino acid sequence derived from a plasmid vector used during production may be added to the amino acid sequence. Examples of such additional sequences include amino acid sequences such as tags, secretion signal peptides, and signal peptides for localization to cell surfaces and organelles. The number of amino acids that can be added is not particularly limited, so long as the protein does not have hydrolytic activity against α-glucosides and has the activity of catalyzing a glycosyltransfer reaction using an activated sugar as a donor substrate, but can be, for example, 1 to 400, 1 to 300, 1 to 200, 1 to 100, 1 to 50, 1 to 40, or 1 to 30.

[0042] In one embodiment, the protein of the present invention has SEQ ID NO: 4 added to the C-terminus of a given amino acid sequence (eg, SEQ ID NO: 3).

[0043] In one embodiment, the protein of the present invention has SEQ ID NO:5 added to the N-terminus of a predetermined amino acid sequence (eg, SEQ ID NO:3 or SEQ ID NO:1). [Table 5] SEQ ID NO: 5 is a sequence that can function as a secretory signal sequence in an expression system using Pichia pastoris. For example, in an expression system using Pichia pastoris, SEQ ID NO: 5 can be cleaved and removed during extracellular secretion, but may remain attached as long as the protein of the present invention does not have hydrolytic activity against α-glucosides and has activity to catalyze a glycosyltransferase reaction using an activated sugar as a donor substrate.

[0044] In one embodiment, the protein of the invention has the amino acid sequence of SEQ ID NO:6. [Table 6]

[0045] In the present disclosure, the method for confirming that a protein and / or enzyme preparation "does not have hydrolytic activity for α-glucosides" is not particularly limited, and can be confirmed by a method commonly used in the art. For example, it can be confirmed by reacting a protein and / or enzyme preparation with maltose to detect the presence or absence of glucose production. Alternatively, an α-glucosidase mutant in which the general acid-base catalytic residue of α-glucosidase is replaced with an amino acid residue that cannot be a general acid-base catalytic residue (e.g., asparagine residue, alanine residue, serine residue, glycine residue, glutamine residue) can be considered to be a protein "not having hydrolytic activity for α-glucosides".

[0046] In the present disclosure, the method for confirming that a protein and / or enzyme preparation "has the activity of catalyzing a transglycosylation reaction using an activated sugar as a donor substrate" is not particularly limited, and can be confirmed, for example, by reacting the protein and / or enzyme preparation with a donor substrate, which is an activated sugar, and an acceptor substrate in an appropriate solvent such as water or alcohol, and checking whether an expected transglycosylation product is detected. If at least one expected transglycosylation product is detected, the protein and / or enzyme preparation "has the activity of catalyzing a transglycosylation reaction using an activated sugar as a donor substrate." When the donor substrate, which is an activated sugar, itself also functions as an acceptor substrate to generate a transglycosylation product, the donor substrate and the acceptor substrate can be the same substance. When a solvent molecule such as water or alcohol also acts as an acceptor substrate to generate a transglycosylation product, the solvent and the acceptor substrate can be the same substance. A specific example is the method of the Examples of the present application (more specifically, Synthesis Reaction Example 8).

[0047] In the present disclosure, the activated sugar is not particularly limited, and any activated sugar commonly used in glycosynthase or glycoligase reactions can be used, but it is preferably a sugar derivative derived from an α-anomeric sugar. Examples include sugar derivatives derived from α-glucose, α-mannose, or α-galactose. More specific examples include sugar derivatives in which the 1-OH of a sugar selected from the group consisting of α-glucose, α-mannose, and α-galactose is substituted with a leaving group (e.g., fluorine, iodine, chlorine, nitrophenol and its derivatives, phosphoric acid, uridine diphosphate, and 4-methylumbelliferone). As a more specific example, Examples of activated sugars derived from α-glucose: 1-fluoro-1-deoxy-α-glucose, 1-bromo-1-deoxy-α-glucose, 1-chloro-1-deoxy-α-glucose, 4-nitrophenyl α-glucoside, 2,4-dinitrophenyl α-glucoside, α-glucose-1-phosphate, α-uridine diphosphate glucoside, 4-methylumbelliferyl α-glucoside, Examples of activated sugars derived from α-mannose: 1-fluoro-1-deoxy-α-mannose, 1-bromo-1-deoxy-α-mannose, 1-chloro-1-deoxy-α-mannose, 4-nitrophenyl α-mannoside, 2,4-dinitrophenyl α-mannoside, α-mannose-1-phosphate, α-uridine diphosphate mannoside, 4-methylumbelliferyl α-mannoside, and Examples of activated sugars derived from α-galactose: 1-fluoro-1-deoxy-α-galactose, 1-bromo-1-deoxy-α-galactose, 1-chloro-1-deoxy-α-galactose, 4-nitrophenyl α-galactoside, 2,4-dinitrophenyl α-galactoside, α-galactose-1-phosphate, α-uridine diphosphate galactoside, and 4-methylumbelliferyl α-galactoside. Examples include: A preferred example of the activated sugar is 1-fluoro-1-deoxy-α-mannose.

[0048] In the present disclosure, since the acceptor substrate specificity is broad, the acceptor substrate is not particularly limited as long as it is a compound that can catalyze a glycosyltransferase reaction by the protein of the present invention, for example, a carboxylic acid compound, a phosphate compound, and a compound having a thiol group. Examples of carboxylic acid compounds include formic acid, acetic acid, butyric acid, octanoic acid, capric acid, lauric acid, palmitic acid, giberic acid, salicylic acid, indole-3-acetic acid, jasmonic acid, abscisic acid, malonic acid, L-lactic acid, glyceric acid, formic acid, oxalic acid, phthalic acid, citric acid, tartaric acid, amino acids, peptides containing aspartic acid / glutamic acid (e.g., angiotensin I), gluconic acid, glucuronic acid, etc., and vinylogues of carboxylic acids such as ascorbic acid are also examples of carboxylic acid compounds. Preferred examples of carboxylic acid compounds include formic acid, acetic acid, butyric acid, octanoic acid, indole-3-acetic acid, jasmonic acid, abscisic acid, malonic acid, L-lactic acid, formic acid, phthalic acid, citric acid, tartaric acid, amino acids, peptides (e.g., angiotensin I), glucuronic acid, and ascorbic acid. Examples of phosphate compounds include pyrophosphate and glucose 6-phosphate. Examples of compounds having a thiol group include cysteine, 2-mercaptoethanol, and 6-deoxy-6-mercaptoglucose.

[0049] In the present disclosure, a transglycosylation product refers to a product obtained by transferring a sugar moiety of an activated sugar, which is a donor substrate, to an acceptor substrate. For example, when the acceptor substrate is a carboxylic acid compound, a sugar ester compound can be obtained. For example, when the acceptor substrate is a phosphate compound, a sugar phosphate compound can be obtained. For example, when the acceptor substrate is a compound having a thiol group, a thioglycoside compound can be obtained. In the present disclosure, the anomeric form of the sugar moiety in the transglycosylation product is not particularly limited, and may be either an α-anomer or a β-anomer. When an activated sugar derived from an α-anomeric sugar is used as a donor substrate, the sugar moiety in the transglycosylation product may be an α-anomeric sugar, but even a β-anomeric transglycosylation product may be within the scope of the present invention.

[0050] In one embodiment, the protein of the present invention and / or the enzymatic agent of the present invention has an activity to catalyze a transglycosylation reaction using, as a donor substrate, an activated sugar derived from a sugar selected from the group consisting of α-glucose, α-mannose, and α-galactose. In this case, the protein of the present invention and / or the enzymatic agent of the present invention may have an activity to catalyze a transglycosylation reaction using, as a donor substrate, an activated sugar other than an activated sugar derived from a sugar selected from the group consisting of α-glucose, α-mannose, and α-galactose.

[0051] In one embodiment, the protein of the present invention and / or the enzymatic preparation of the present invention has an activity of catalyzing a transglycosylation reaction using an activated sugar derived from a sugar selected from the group consisting of α-mannose and α-galactose as a donor substrate. In this case, the protein of the present invention and / or the enzymatic preparation of the present invention may have an activity of catalyzing a transglycosylation reaction using an activated sugar other than an activated sugar derived from a sugar selected from the group consisting of α-mannose and α-galactose as a donor substrate.

[0052] In one embodiment, the protein of the present invention and / or the enzymatic agent of the present invention has an activity to catalyze a transglycosylation reaction using an activated sugar derived from α-mannose as a donor substrate. In this case, the protein of the present invention and / or the enzymatic agent of the present invention may have an activity to catalyze a transglycosylation reaction using an activated sugar other than an activated sugar derived from α-mannose as a donor substrate.

[0053] In one embodiment, the protein of the present invention and / or the enzyme preparation of the present invention has an activity of catalyzing a transglycosylation reaction using an activated sugar derived from 1-fluoro-1-deoxy-α-mannose as a donor substrate. In this case, the protein of the present invention and / or the enzyme preparation of the present invention may have an activity of catalyzing a transglycosylation reaction using an activated sugar other than an activated sugar derived from 1-fluoro-1-deoxy-α-mannose as a donor substrate.

[0054] In one embodiment, the protein of the present invention and / or the enzyme preparation of the present invention are used to catalyze a transglycosylation reaction with a donor substrate that is an activated sugar (e.g., derived from a sugar selected from the group consisting of α-glucose, α-mannose, and α-galactose (preferably α-mannose and α-galactose, more preferably α-mannose)).

[0055] In one embodiment, the protein of the present invention and / or the enzyme preparation of the present invention are used to transfer a sugar from a donor substrate, which is an activated sugar (e.g., derived from a sugar selected from the group consisting of α-glucose, α-mannose, and α-galactose (preferably α-mannose and α-galactose, more preferably α-mannose), to an acceptor substrate.

[0056] In one embodiment, the protein of the present invention and / or the enzyme preparation of the present invention are used to produce a transglycosylation product by reacting a donor substrate, which is an activated sugar (e.g., derived from a sugar selected from the group consisting of α-glucose, α-mannose, and α-galactose (preferably α-mannose and α-galactose, more preferably α-mannose), with an acceptor substrate.

[0057] The transglycosylation products obtained using the protein of the present invention and / or the enzyme preparation of the present invention can be used for the production of foods, feeds, cosmetics, reagents, or pharmaceuticals, depending on their properties.

[0058] In the present disclosure, the reaction conditions between a donor substrate and an acceptor substrate in the presence of the protein of the present invention and / or the enzyme preparation of the present invention are not particularly limited as long as they are conditions under which a glycosyltransfer reaction can occur, and can be appropriately selected depending on the protein of the present invention and / or the enzyme preparation of the present invention, the donor substrate, and the acceptor substrate used. In the present disclosure, when a donor substrate, which is an activated sugar, itself acts as an acceptor substrate to generate a transglycosylation product, the donor substrate and the acceptor substrate can be the same substance.

[0059] The reaction is preferably carried out in a liquid. The type of liquid component is not particularly limited as long as it is a liquid component that allows the transglycosylation reaction to proceed, but it is preferable that the liquid component is, for example, a liquid component in which both substrates and the enzyme can be dissolved so that they can easily come into contact with each other. In cases where the liquid component also serves as the acceptor substrate to produce a transglycosylation product, the solvent and the acceptor substrate can be the same substance.

[0060] The reaction can be carried out under conditions of, for example, pH 3 to 8, temperature 10° C. to 45° C., and / or reaction time 1 minute to 300 hours. Examples of the pH include pH 3.5 to 6.5, pH 4 to 6.5, or pH 5.5 to 6.5. Examples of the reaction temperature include 15°C to 40°C, 25°C to 39°C, and 30°C to 38°C. Examples of the reaction time include 1 minute to 200 hours, 1 minute to 100 hours, and 20 minutes to 100 hours.

[0061] In one aspect, the present application provides a method for producing a protein of the present invention, comprising culturing the host cell described above and recovering the protein of the present invention from the culture.

[0062] The method for producing the protein of the present invention is not particularly limited, and the protein can be produced by a conventional method. The protein of the present invention may be produced by a microorganism such as an Aspergillus bacterium, may be a protein synthesized by chemical synthesis, or may be a recombinant protein produced by gene recombination technology. For example, the protein of the present application can be obtained by obtaining a polynucleotide encoding the protein of the present invention from a polynucleotide encoding a specific α-glucosidase by a site-specific mutagenesis method such as a method combining the Kunkel method and PCR method (e.g., Inverse PCR, QuikChange method), incorporating the polynucleotide into a vector such as a plasmid, bacteriophage, cosmid, etc., by a conventional method, transforming a host cell with the vector, culturing the transformed cell, and purifying the transformed cell from the culture as appropriate. Alternatively, the protein of the present application can be obtained by introducing a mutation into a polynucleotide encoding a specific α-glucosidase encoded in the genomic DNA of a bacterium of the genus Aspergillus or the like by genome editing (e.g., CRISPR-Cas9) to convert the polynucleotide into a polynucleotide encoding the protein of the present invention, culturing the genome-edited bacterium of the genus Aspergillus, and purifying the transformed cell from the culture as appropriate.

[0063] In one aspect, the present application provides a polynucleotide encoding a protein of the present invention. In one embodiment, the present application provides a polynucleotide comprising (or consisting of) SEQ ID NO:7, 8, or 9. SEQ ID NO:7 is an example of a polynucleotide that encodes the amino acid sequence of SEQ ID NO:1. SEQ ID NO:8 is an example of a polynucleotide that encodes the amino acid sequence of SEQ ID NO:3. SEQ ID NO:9 is an example of a polynucleotide that encodes the amino acid sequence of SEQ ID NO:6. TIFF2024009555000008.tif193155 TIFF2024009555000009.tif185155 TIFF2024009555000010.tif213155

[0064] In one aspect, the present application provides a vector comprising the polynucleotide. The vector is preferably an expression vector. For example, a commonly used expression vector can be appropriately selected and used.

[0065] In one aspect, the present application provides a host cell transformed with the vector. An agent containing a host cell can be included as an embodiment of the enzyme agent of the present invention. The host cell is not particularly limited, and can be a microbial cell such as a bacterium, yeast, filamentous fungus, or actinomycete, a plant cell, or an animal cell.

[0066] In this disclosure, unless otherwise specified, "%", "parts", etc. are by weight and numerical ranges are stated as including their endpoints. EXAMPLES

[0067] The present invention will be described more specifically based on the following examples, but the present invention is not limited to these examples.

[0068] Preparation of enzyme: The ANG_D660N recombinant enzyme (SEQ ID NO: 1) was produced using Pichia pastoris GS115 strain as a heterologous host. The plasmid vector used was pGAPZαA from ThermoFischer Scientific, and ANG_D660N was produced as an extracellular secretory protein according to the attached protocol. In addition, a protein (sequence number 6) having a secretory signal sequence added to the N-terminus of ANG_D660N is produced in the heterologous host, and the secretory signal sequence is cleaved and removed upon secretion outside the bacterial cells, resulting in secretion of ANG_D660N (sequence number 1) outside the bacterial cells. The nucleotide sequence encoding the ANG_D660N recombinase (SEQ ID NO:1) is shown in SEQ ID NO:7. SEQ ID NO:9 shows the nucleotide sequence encoding the protein (SEQ ID NO:6) in which a secretory signal sequence has been added to the N-terminus of ANG_D660N. ANG_D660N was precipitated from the P. pastoris culture supernatant by ammonium sulfate precipitation and then purified by Ni-Chelating Sepharose FastFlow column chromatography until it became electrophoretically homogeneous. The purified enzyme was dialyzed against 20 mM sodium acetate buffer (pH 5.0), and the appropriately concentrated enzyme solution was used for the synthesis reaction.

[0069] Preparation of glycosyl donors: 1-Fluoro-1-deoxy-α-mannose, 1-fluoro-1-deoxy-α-glucose, and 1-fluoro-1-deoxy-α-galactose were synthesized according to a previously reported method (DOI: 10.1246 / cl.1984.1747).

[0070] Synthesis reaction example 1 (reaction with short-chain fatty acid): Formic acid, acetic acid or butyric acid was titrated with sodium hydroxide to pH 4.0. 3.12 uM ANG_D660N, 25 mM 1-fluoro-1-deoxy-α-mannose and 40 mM formic acid, acetic acid or butyric acid (pH 4.0) were mixed and kept at 37°C for 24 to 72 hours. As a negative control, a reaction was also performed in which ANG_D660N was replaced with 4 mM sodium acetate buffer (pH 5.0). The reaction solution after the time elapsed was analyzed by TLC. The results are shown in Figure 1. When formic acid, acetic acid and butyric acid were used, products not seen in the negative control were detected. As a result of ESI-MS analysis of the reaction products with formic acid, acetic acid and butyric acid, molecular ion-related peaks estimated to be derived from 1-O-mannose formate, 1-O-acetylmannose and 1-O-mannose butyrate were detected (Figures 2 to 4). Sodium hydroxide was titrated into acetic acid to pH 4.0, 5.0, or 6.0. 3.12 uM ANG_D660N, 25 mM 1-fluoro-1-deoxy-α-mannose, and 40 mM acetic acid (pH 4.0, 5.0, or 6.0) were mixed and kept at 37°C for 1 to 72 hours, and the product was analyzed by TLC over time. The results are shown in Figure 5. In the reaction at pH 6.0, the spot of 1-fluoro-1-deoxy-α-mannose disappeared most quickly, and the spot of the product estimated to be 1-O-acetylmannose showed the strongest intensity. The disappearance of 1-fluoro-1-deoxy-α-mannose was observed at all pH values. The amount of mannose in the reaction solution at 72 hours after the reaction was quantified using a D-Mannose / D-Fructose / D-Glucose Assay kit (Megazyme) (Table 6). The respective amounts of mannose were 20.7±0.3 mM (pH 4.0), 19.2±0.1 mM (pH 5.0), and 18.6±0.1 mM (pH 6.0). Based on the TLC results, the reaction products were only mannose and a product estimated to be 1-O-acetylmannose, and the sum of the two was equal to the substrate concentration (25 mM), based on which the concentration of the transfer product was calculated. The estimated concentrations and yields of the transfer product at each pH were 4.3 mM (17.2%, pH 4.0), 5.8 mM (23.1%, pH 5.0), and 6.4 mM (25.6%, pH 6.0). [Table 6]

[0071] Synthesis reaction example 2 (reaction with medium-chain and long-chain fatty acids): Octanoic acid (C8), capric acid (C10) or lauric acid (C12) was dissolved in ethanol to a concentration of 500 mM. Palmitic acid (C16) was dissolved in diethyl ether to a concentration of 500 mM. A 500 mM fatty acid solution was mixed in a volume of 1 / 5 to a reaction solution consisting of 3.12 uM ANG_D660N, 25 mM 1-fluoro-1-deoxy-α-mannose and 40 mM sodium acetate buffer (pH 4.0) and kept at 37°C for 72 hours. As a negative control, a reaction was also carried out in which ANG_D660N was replaced with 4 mM sodium acetate buffer (pH 5.0). The reaction solution after the time had passed was analyzed by TLC. The results are shown in Figure 1. When octanoic acid was used, a product was detected that was not seen in the negative control. As a result of ESI-MS analysis of the reaction product with octanoic acid, a molecular ion-related peak presumably derived from 1-O-mannose octanoate was detected (Figure 6).

[0072] Synthesis reaction example 3 (reaction with plant hormone): Ascorbic acid was dissolved in water to a concentration of 500 mM. Gibberellic acid, salicylic acid, indole-3-acetic acid, jasmonic acid or abscisic acid was dissolved in ethanol to a concentration of 500 mM. A reaction solution consisting of 3.12 uM ANG_D660N, 25 mM 1-fluoro-1-deoxy-α-mannose and 40 mM sodium acetate buffer (pH 4.0) was mixed with 1 / 5 the amount of 500 mM plant hormone solution and kept at 37°C for 72 hours. As a negative control, a reaction was also carried out in which ANG_D660N was replaced with 4 mM sodium acetate buffer (pH 5.0). The reaction solution after the time had passed was analyzed by TLC. The results are shown in Figure 7. When ascorbic acid, indole-3-acetic acid, jasmonic acid or abscisic acid was used, products that were not seen in the negative control were detected. ESI-MS analysis of the reaction products with ascorbic acid, indole acetic acid, jasmonic acid, or abscisic acid detected molecular ion-related peaks presumably derived from compounds formed by condensation of mannose with plant hormones (Figures 8 to 11). Salicylic acid was dissolved in an aqueous solution of sodium hydroxide, and then the pH was adjusted to 4.0 by titration with hydrochloric acid, and the concentration was diluted to 100 mM. A reaction solution consisting of 3.12 uM ANG_D660N, 25 mM 1-fluoro-1-deoxy-α-mannose, and 40 mM salicylic acid-Na (pH 4.0) was kept at 37°C for 72 hours. As a negative control, a reaction was also performed in which ANG_D660N was replaced with 4 mM sodium acetate buffer (pH 5.0). The reaction solution after the time elapsed was analyzed by TLC. The results are shown in Figure 12. In the enzyme reaction, a product was detected that was not seen in the negative control. As a result of ESI-MS analysis of the reaction product, a molecular ion-related peak was detected that was presumed to be derived from a compound in which mannose and salicylic acid were condensed (Figure 13).

[0073] Synthesis reaction example 4 (reaction with organic acid): Malonic acid, L-lactic acid, glyceric acid, formic acid, oxalic acid, pyrophosphoric acid, phthalic acid, citric acid or tartaric acid were titrated with sodium hydroxide to pH 4.0. 3.12 uM ANG_D660N, 25 mM 1-fluoro-1-deoxy-α-mannose and 40 mM organic acid solution (pH 4.0) were mixed and kept at 37°C for 0.5 to 96 hours. As a negative control, a reaction was also carried out in which ANG_D660N was replaced with 4 mM sodium acetate buffer (pH 5.0). The reaction solution after the time elapsed was analyzed by TLC. The results are shown in Figures 14 and 15. When malonic acid, L-lactic acid, formic acid, pyrophosphoric acid, phthalic acid, citric acid or tartaric acid was used, products not seen in the negative control were detected. As a result of ESI-MS analysis of the reaction products with malonic acid, L-lactic acid, glyceric acid, pyrophosphoric acid, phthalic acid, citric acid, or tartaric acid, molecular ion-related peaks presumably derived from compounds formed by condensation of mannose and phthalic acid were detected (Figures 16 to 22).

[0074] Synthesis reaction example 5 (reaction with amino acids): 3.12 uM ANG_D660N, 25 mM 1-fluoro-1-deoxy-α-mannose, and 100 mM amino acids (serine (Ser), threonine (Thr), cysteine ​​(Cys) or cysteine ​​methyl ester (CysMe)) were mixed and kept at 37°C for 0.5 to 96 hours. As a negative control, a reaction was also performed in which ANG_D660N was replaced with 4 mM sodium acetate buffer (pH 5.0). The reaction solution after the time elapsed was analyzed by TLC. The results are shown in Figure 23. When serine, threonine, or cysteine ​​was used, products not seen in the negative control were detected. As a result of ESI-MS analysis of the reaction products, molecular ion-related peaks estimated to be derived from compounds in which mannose and each amino acid were condensed were detected (Figures 24 to 26).

[0075] Synthesis reaction example 6 (reaction with peptide): 3.12 uM ANG_D660N, 25 mM 1-fluoro-1-deoxy-α-mannose, and 0.5 mg / mL angiotensin I (DRVYIHPFHL) were mixed and kept at 37°C for 72 hours. As a negative control, a reaction was also carried out in which ANG_D660N was replaced with 4 mM sodium acetate buffer (pH 5.0). The reaction solution after the time had elapsed was analyzed by LC-MS. A peak was detected in the enzyme reaction solution that was not seen in the negative control, and the composition formula (C 68 H 99 N 17 O 19 ) matched the formula of a compound formed by condensation of mannose and angiotensin I (Figure 27).

[0076] Synthesis reaction example 7 (reaction with glucose derivative carboxylic acid): 3.12 uM ANG_D660N, 25 mM 1-fluoro-1-deoxy-α-mannose, and 100 mM gluconic acid or glucuronic acid were mixed and kept at 37°C for 48 hours. As a negative control, a reaction was also carried out in which ANG_D660N was replaced with 4 mM sodium acetate buffer (pH 5.0). The reaction solution after the time had elapsed was analyzed by TLC. The results are shown in Figure 28. When glucuronic acid was used, a product was detected that was not seen in the negative control.

[0077] Synthesis reaction example 8 (reaction using 1-fluoro-1-deoxy-α-glucose and 1-fluoro-1-deoxy-α-galactose as sugar donors): Acetic acid was titrated with sodium hydroxide to pH 4.0. 3.12 uM ANG_D660N, 25 mM 1-fluoro-1-deoxy-α-glucose or 1-fluoro-1-deoxy-α-galactose, and 40 mM acetic acid (pH 4.0) were mixed and kept at 37°C for 0.5 to 72 hours. As a negative control, a reaction was also carried out in which ANG_D660N was replaced with 4 mM sodium acetate buffer (pH 5.0). The reaction solution after the time elapsed was analyzed by TLC. The results are shown in Figures 29 and 30. In the enzyme reactions using either 1-fluoro-1-deoxy sugar as a sugar donor, products were detected that were not seen in the negative control.

[0078] As verified by the synthetic reaction examples above, ANG_D660N was shown to catalyze glycosyltransferase reactions using 1-fluoro-1-deoxy-α-mannose, 1-fluoro-1-deoxy-α-glucose, or 1-fluoro-1-deoxy-α-galactose as glycosyl donors and various carboxylic acid compounds and phosphate compounds as glycosyl acceptors. In the above synthesis reaction examples, there are some glycosyl donors for which no reaction products were detected. However, this does not simply mean that no reaction products were detected under the reaction conditions of the synthesis reaction example, and it is possible that the reaction products can be obtained by changing the reaction conditions. [Industrial Applicability]

[0079] The present invention is useful for producing transglycosylation products.

Claims

1. A protein comprising any one of the following amino acid sequences (a) to (g), which does not have hydrolytic activity against α-glucosides and has the activity of catalyzing a transglycosylation reaction using an activated sugar as a donor substrate: (a) an amino acid sequence in which the amino acid residue at position 660 in the amino acid sequence of SEQ ID NO: 2 has been substituted; (b) an amino acid sequence in which the amino acid residue at position 660 in the amino acid sequence of SEQ ID NO: 2 has been substituted and one or more amino acid residues have been further substituted, deleted, inserted and / or added; (c) an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, wherein the amino acid residue at the position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is an amino acid residue other than an aspartic acid residue; (d) an amino acid sequence of an α-glucosidase having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, in which the amino acid residue at the position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is substituted; (e) an amino acid sequence of an α-glucosidase having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, in which the amino acid residue at the position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 has been substituted, and further in which one or more amino acid residues have been substituted, deleted, inserted, and / or added; (f) an amino acid sequence of an α-glucosidase having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, in which a general acid-base catalytic residue has been substituted; (g) An amino acid sequence of an α-glucosidase having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO: 2, in which a general acid-base catalytic residue has been substituted and one or more amino acid residues have been substituted, deleted, inserted and / or added.

2. A protein comprising any one of the following amino acid sequences (a) to (g), which does not have hydrolytic activity against α-glucosides and has the activity of catalyzing a transglycosylation reaction using an activated sugar as a donor substrate: (a) an amino acid sequence in which the amino acid residue at position 660 in the amino acid sequence of SEQ ID NO: 2 is substituted with an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues; (b) an amino acid sequence in which the amino acid residue at position 660 in the amino acid sequence of SEQ ID NO: 2 is substituted with an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues, and in which one or more amino acid residues are further substituted, deleted, inserted, and / or added; (c) an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, wherein when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2, the amino acid residue at the position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 is an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues; (d) an amino acid sequence of an α-glucosidase having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, in which the amino acid residue at the position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2 is substituted with an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues; (e) an amino acid sequence of an α-glucosidase having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, in which, when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 2, the amino acid residue at the position corresponding to position 660 in the amino acid sequence of SEQ ID NO: 2 is substituted with an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues, and in which one or more amino acid residues are further substituted, deleted, inserted, and / or added; (f) an amino acid sequence of an α-glucosidase having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, in which the general acid-base catalytic residue is substituted with an amino acid residue selected from the group consisting of asparagine residue, alanine residue, serine residue, glycine residue, and glutamine residue; (g) an amino acid sequence of an α-glucosidase having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, in which a general acid-base catalytic residue is substituted with an amino acid residue selected from the group consisting of asparagine residue, alanine residue, serine residue, glycine residue, and glutamine residue, and in which one or more amino acid residues are further substituted, deleted, inserted, and / or added. The protein of claim 1 ,

3. The protein of claim 1, comprising the amino acid sequence of SEQ ID NO:

3.

4. A protein comprising any one of the following amino acid sequences (a) to (c), which does not have hydrolytic activity against α-glucosides and has the activity of catalyzing a transglycosylation reaction using an activated sugar as a donor substrate: (a) the amino acid sequence of SEQ ID NO:3; (b) an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted and / or added in the amino acid sequence of SEQ ID NO: 3, and when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 3, the amino acid residue at the position corresponding to position 635 in the amino acid sequence of SEQ ID NO: 3 is an amino acid residue other than an aspartic acid residue; (c) An amino acid sequence having a sequence identity of 60% or more with the amino acid sequence of SEQ ID NO: 3, wherein when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 3, the amino acid residue at the position corresponding to position 635 in the amino acid sequence of SEQ ID NO: 3 is an amino acid residue other than an aspartic acid residue.

5. A protein comprising any one of the following amino acid sequences (a) to (c), which does not have hydrolytic activity against α-glucosides and has the activity of catalyzing a transglycosylation reaction using an activated sugar as a donor substrate: (a) the amino acid sequence of SEQ ID NO:3; (b) an amino acid sequence in which one or more amino acid residues are substituted, deleted, inserted, and / or added in the amino acid sequence of SEQ ID NO: 3, and when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 3, the amino acid residue at the position corresponding to position 635 in the amino acid sequence of SEQ ID NO: 3 is an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues; (c) an amino acid sequence having 60% or more sequence identity with the amino acid sequence of SEQ ID NO: 3, wherein the amino acid residue at the position corresponding to position 635 in the amino acid sequence of SEQ ID NO: 3 when the amino acid sequence is aligned with the amino acid sequence of SEQ ID NO: 3 is an amino acid residue selected from the group consisting of asparagine residues, alanine residues, serine residues, glycine residues, and glutamine residues. The protein of claim 4,

6. 2. The protein of claim 1, wherein the activated sugar is derived from a sugar selected from the group consisting of α-glucose, α-mannose, and α-galactose.

7. The activated sugar is 1-fluoro-1-deoxy-α-glucose, 1-bromo-1-deoxy-α-glucose, 1-chloro-1-deoxy-α-glucose, 4-nitrophenyl α-glucoside, 2,4-dinitrophenyl α-glucoside, α-glucose-1-phosphate, α-uridine diphosphate glucoside, 4-methylumbelliferyl α-glucoside, 1-fluoro-1-deoxy-α-mannose, 1-bromo-1-deoxy-α-mannose, 1-chloro-1-deoxy-α-mannose, 4-nitrophenyl α-mannoside, 2,4-dinitrophenyl α-mannoside, α-mannose-1-phosphate, α-uridine diphosphate mannoside, 4-methylumbelliferyl α-mannoside, 1-fluoro-1-deoxy-α-galactose, 1-bromo-1-deoxy-α-galactose, 1-chloro-1-deoxy-α-galactose, 4-nitrophenyl α-galactoside, 2,4-dinitrophenyl α-galactoside, α-galactose-1-phosphate, α-uridine diphosphate galactoside, and 4-Methylumbelliferyl α-galactoside The protein of claim 1, selected from the group consisting of:

8. 2. The protein of claim 1, wherein the activated sugar is derived from a sugar selected from the group consisting of α-mannose and α-galactose.

9. The protein according to claim 1, wherein the α-glucosidase is an α-glucosidase classified into Glycoside Hydrolase Family 31.

10. The protein according to claim 1, wherein the α-glucosidase is derived from the genus Aspergillus, Sulfolobus, Schwanniomyces, Saccharomyces, Schizosaccharomyces, Bacteroides, Acremonium, or Podospora.

11. The protein of claim 1, consisting of the amino acid sequence of SEQ ID NO:

1.

12. 2. The protein of claim 1, wherein the acceptor substrate in the glycosyltransfer reaction is a compound selected from the group consisting of a carboxylic acid compound, a phosphate compound, and a compound having a thiol group.

13. An enzyme preparation comprising the protein according to any one of claims 1 to 12.

14. The enzyme preparation according to claim 13, for catalyzing a transglycosylation reaction using, as a donor substrate, an activated sugar derived from a sugar selected from the group consisting of α-mannose and α-galactose.

15. A polynucleotide encoding the protein according to any one of claims 1 to 12.

16. A vector comprising the polynucleotide of claim 15.

17. A host cell transformed with the vector of claim 16.

18. A method for producing a protein described in any one of claims 1 to 12, comprising culturing a host cell transformed with a vector containing a polynucleotide encoding the protein described in any one of claims 1 to 12, and recovering the protein described in any one of claims 1 to 12 from the culture.

19. A method for transferring a sugar from a donor substrate to an acceptor substrate, the method comprising a step of reacting a donor substrate that is an activated sugar with an acceptor substrate in the presence of the protein according to any one of claims 1 to 12.

20. The method of claim 19, wherein the activated sugar is derived from a sugar selected from the group consisting of α-glucose, α-mannose, and α-galactose.

21. The activated sugar is 1-fluoro-1-deoxy-α-glucose, 1-bromo-1-deoxy-α-glucose, 1-chloro-1-deoxy-α-glucose, 4-nitrophenyl α-glucoside, 2,4-dinitrophenyl α-glucoside, α-glucose-1-phosphate, α-uridine diphosphate glucoside, 4-methylumbelliferyl α-glucoside, 1-fluoro-1-deoxy-α-mannose, 1-bromo-1-deoxy-α-mannose, 1-chloro-1-deoxy-α-mannose, 4-nitrophenyl α-mannoside, 2,4-dinitrophenyl α-mannoside, α-mannose-1-phosphate, α-uridine diphosphate mannoside, 4-methylumbelliferyl α-mannoside, 1-fluoro-1-deoxy-α-galactose, 1-bromo-1-deoxy-α-galactose, 1-chloro-1-deoxy-α-galactose, 4-nitrophenyl α-galactoside, 2,4-dinitrophenyl α-galactoside, α-galactose-1-phosphate, α-uridine diphosphate galactoside, and 4-Methylumbelliferyl α-galactoside 20. The method of claim 19, selected from the group consisting of:

22. The method of claim 19, wherein the activated sugar is derived from a sugar selected from the group consisting of α-mannose and α-galactose.

23. The method of claim 19, wherein the receptor substrate is a compound selected from the group consisting of carboxylic acid compounds, phosphate compounds, and compounds having a thiol group.

24. A method for producing a transglycosylation product, comprising the step of reacting a donor substrate, which is an activated sugar, with an acceptor substrate in the presence of the protein according to any one of claims 1 to 12.

25. 25. The method of claim 24, wherein the activated sugar is derived from a sugar selected from the group consisting of α-glucose, α-mannose, and α-galactose.

26. The activated sugar is 1-fluoro-1-deoxy-α-glucose, 1-bromo-1-deoxy-α-glucose, 1-chloro-1-deoxy-α-glucose, 4-nitrophenyl α-glucoside, 2,4-dinitrophenyl α-glucoside, α-glucose-1-phosphate, α-uridine diphosphate glucoside, 4-methylumbelliferyl α-glucoside, 1-fluoro-1-deoxy-α-mannose, 1-bromo-1-deoxy-α-mannose, 1-chloro-1-deoxy-α-mannose, 4-nitrophenyl α-mannoside, 2,4-dinitrophenyl α-mannoside, α-mannose-1-phosphate, α-uridine diphosphate mannoside, 4-methylumbelliferyl α-mannoside, 1-fluoro-1-deoxy-α-galactose, 1-bromo-1-deoxy-α-galactose, 1-chloro-1-deoxy-α-galactose, 4-nitrophenyl α-galactoside, 2,4-dinitrophenyl α-galactoside, α-galactose-1-phosphate, α-uridine diphosphate galactoside, and 4-Methylumbelliferyl α-galactoside 25. The method of claim 24, selected from the group consisting of:

27. 25. The method of claim 24, wherein the activated sugar is derived from a sugar selected from the group consisting of α-mannose and α-galactose.

28. 25. The method of claim 24, wherein the acceptor substrate is a compound selected from the group consisting of a carboxylic acid compound, a phosphate compound, and a compound having a thiol group.

29. 25. A method for producing a food, feed, cosmetic, reagent, or pharmaceutical, comprising the steps of obtaining a transglycosylation product by the method of claim 24 and obtaining a food, feed, cosmetic, or pharmaceutical using the transglycosylation product obtained in said step.

Images