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WO2019035482A1 - Protéine présentant une activité d'épimérisation - Google Patents

Protéine présentant une activité d'épimérisation Download PDF

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Publication number
WO2019035482A1
WO2019035482A1 PCT/JP2018/030497 JP2018030497W WO2019035482A1 WO 2019035482 A1 WO2019035482 A1 WO 2019035482A1 JP 2018030497 W JP2018030497 W JP 2018030497W WO 2019035482 A1 WO2019035482 A1 WO 2019035482A1
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amino acid
acid sequence
seq
identity
protein
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Japanese (ja)
Inventor
亘 佐分利
春英 森
貴久 飯塚
藤本　佳則
宏基 高木
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Hokkaido University NUC
Japan Maize Products Co Ltd
Nihon Shokuhin Kako Co Ltd
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Hokkaido University NUC
Japan Maize Products Co Ltd
Nihon Shokuhin Kako Co Ltd
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Publication of WO2019035482A1 publication Critical patent/WO2019035482A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/90—Isomerases (5.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides

Definitions

  • the present invention relates to a protein having epimerization activity.
  • This application claims the priority of Japanese Patent Application No. 2017-157664 filed on Aug. 17, 2017, the entire description of which is incorporated herein by reference in particular.
  • Mannose is an isomer of glucose, and is well known as glucomannan extracted from konjac etc., hemicellulose, or constituent sugars of sugar chains present on cell surfaces etc. Mannose itself is known to be contained in cranberries.
  • mannose mannose binding protein
  • Patent Document 1 discloses a technology relating to a harmful bacterial infection inhibitor or a harmful bacterial infection preventing feed containing a mannose-containing sugar composition as an active ingredient.
  • Patent Document 2 describes a food texture improver for food and drink containing mannose as an active ingredient.
  • the first chemical reaction method is a method of producing sorbitol and mannitol by reducing fructose, fractionating mannitol, and further, preparing mannose by oxidizing mannitol.
  • this method is problematic in terms of efficiency because it requires multistep steps and catalytic reaction at high temperature and pressure.
  • the second chemical reaction method is a method of preparing mannose by hydrolyzing glucomannan and mannan.
  • this method has the problem that the substrate is expensive and the preparation of the substrate is laborious.
  • the third chemical reaction method is a method of hydrolyzing hemicellulose such as copra meal.
  • this method has a problem that the yield of mannose is poor because the constituent sugars are various, and in the case of acid hydrolysis, the yield is further degraded and the purification load is increased.
  • the fourth chemical reaction method is a method of preparing mannose by isomerizing glucose under alkaline conditions.
  • this method has a problem that the yield is poor due to the decomposition of sugar and the purification load is large.
  • the fifth chemical reaction method is also a method of preparing mannose by isomerizing fructose under alkaline conditions.
  • this method is problematic in terms of yield and purification load, and fructose is relatively expensive compared to glucose, and fructose remains in the reaction system and mannose There is also a problem in that it is difficult to separate them.
  • the first enzyme method is a method of preparing mannose by causing mannose isomerase to act on fructose.
  • mannose isomerase to act on fructose.
  • the second enzymatic method is a method of preparing mannose by reacting glucose or fructose with aldose-ketose isomerase (hereinafter referred to as "AKI").
  • AKI aldose-ketose isomerase
  • the hydroxyl group at the 2-position of the reducing terminal sugar residue is 2-epimerized Cellobiose 2-epimerase (Cellobiose 2-epimerase, hereinafter referred to as “CE”) is known as an enzyme catalyzing the (isomerization) reaction or its reverse reaction (Patent Document 3, Patent Document 4).
  • the enzyme can convert cellobiose in which glucose is ⁇ -1,4 linked to 4-O- ⁇ -glucosylmannose or reverse reaction thereof.
  • Patent Document 4 describes that mannose is obtained when the cellobiose 2-epimerase is caused to act on glucose (third enzyme method).
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2001-213,781
  • Patent Document 2 Japanese Patent Laid-Open Application No. 2001-275583
  • Patent Document 3 Japanese Patent Laid-Open Application No. 2012-130332
  • Patent Document 4 WO2010 / 090095
  • An object of the present invention is to provide a means for producing mannose more inexpensively and easily. More specifically, it is an object of the present invention to provide a method capable of efficiently producing mannose using relatively inexpensive raw materials and an enzyme or enzyme preparation that can be used for the method. It is also an object of the present invention to provide a method capable of efficiently producing talose and an enzyme or enzyme preparation that can be used for the method.
  • the present inventors have searched for isomerization enzymes of various sugars including CE.
  • Runella slithyformis has three genes presumed to encode N-acylglucosamine 2-epimerase (N-acylglucosamine 2-epimerase, hereinafter referred to as "AGE") on the genome Known (Gene Bank).
  • N-acylglucosamine 2-epimerase hereinafter referred to as "AGE”
  • AGE N-acylglucosamine 2-epimerase
  • ME mannose 2-epimerase
  • These proteins have a specific insertion sequence as compared to known primary amino acid sequences such as CE, and the presence of the insertion sequence is extremely important in terms of function as an ME, and further similar to the insertion sequence.
  • the present invention has been completed by newly finding a plurality of MEs.
  • the present invention is as follows. [1] (A) has an insertion sequence of an amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2, An amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 on the N terminal side of the inserted sequence, and SEQ ID NO: 2 on the C terminal side of the inserted sequence A protein having an amino acid sequence of 50% or more identity to the amino acid sequence of the 264 to 423 amino acid residue of the amino acid sequence shown in and having an epimerylation activity of glucose and mannose, or (b) SEQ ID NO: 12 Has an insertion sequence of an amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown; An amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO:
  • An amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2 is General formula (1): X 1 APQIKFDX 2 X 3 WGWDRFX 4 X 5 X 6 X 7 X 8 X 9 X 10 (where X 1 is V or I, X 2 is I or V, X 3 is V or I, X 4 is N, T or S, X 5 is P or E, X 6 is D or G, X 7 is G or D, X 8 is L, V, Q or A , X 9 is K or Q and X 10 is S, A, E or K) An amino acid sequence having an identity of 90% or more with the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2; An amino acid sequence having at least 90% identity to the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 4
  • An amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 is An amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2; An amino acid sequence having an identity of 90% or more with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 4; An amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 6; Amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 8 or the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10 Amino acid sequence with 90% or more identity with An amino acid sequence having 50% or more identity to the amino acid sequence of the 264 to 423 amino acid residue of the amino acid sequence
  • An amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 is General formula (2): KX 11 PAINIX 12 RTWNAEREX 13 X 14 EX 15 ID (Wherein X 11 is L or I, X 12 is K or Y, X 13 is A or T, X 14 is G or N, and X 15 is K or R) Array, Amino acid sequence having at least 90% identity to the amino acid sequence of amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 or amino acid sequence of amino acid residue 246 to 267 of the amino acid sequence shown in SEQ ID NO: 14 The protein according to [1], which is an amino acid sequence having an identity of 90% or more.
  • An amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 is Amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence of 1 to 245 amino acid residues of the amino acid sequence shown in SEQ ID NO: 14
  • An amino acid sequence having 50% or more identity to the amino acid sequence of the 263-419 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 is Amino acid sequence having at least 90% identity with the amino acid sequence of 263-419 amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence of 268-430 amino acid residues of the amino acid sequence shown in SEQ ID NO: 14
  • An epimerising agent for glucose and mannose comprising the protein according to any one of [1] to [5] as an active ingredient.
  • [8] (C) has an insertion sequence of an amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 264 of the amino acid sequence shown in SEQ ID NO: 10, An amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10 at the N-terminal side of the inserted sequence, and SEQ ID NO: 10 at the C-terminal side of the inserted sequence A protein having an amino acid sequence of 50% or more identity to the amino acid sequence of the 265 to 428 amino acid residue of the amino acid sequence shown in and having an epimerylation activity of galactose and talose, or (b) SEQ ID NO: 12 Has an insertion sequence of an amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown; An amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ
  • An amino acid sequence having an identity of 70% or more with the amino acid sequence of 241 to 264 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10 is an amino acid sequence of 241 to 264 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10
  • Amino acid sequence with 90% or more identity with An amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10 is An amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10
  • An amino acid sequence having an identity of 50% or more with the amino acid sequence of 265 to 428 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10 is an amino acid sequence of 265 to 428 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10
  • An amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 is General formula (2): KX 11 PAINIX 12 RTWNAEREX 13 X 14 EX 15 ID (Wherein X 11 is L or I, X 12 is K or Y, X 13 is A or T, X 14 is G or N, and X 15 is K or R) Array, Amino acid sequence having at least 90% identity to the amino acid sequence of amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 or amino acid sequence of amino acid residue 246 to 267 of the amino acid sequence shown in SEQ ID NO: 14 Amino acid sequence with 90% or more identity with An amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 is Amino acid sequence having 90% or more identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence
  • a glucose and mannose epimerizing agent comprising a protein having an amino acid sequence and having a glucose and mannose epimerization activity as an active ingredient.
  • amino acid sequence of the known or unknown protein has 70% or more identity with the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 12 Inserting an amino acid sequence having an identity of 70% or more with the amino acid sequence of 241 to 262 amino acid residues of the amino acid sequence, or (e) at least a partial sequence of the amino acid sequence of the known or unknown amino acid sequence.
  • an amino acid sequence having an identity of 70% or more is General formula (1): X 1 APQIKFDX 2 X 3 WGWDRFX 4 X 5 X 6 X 7 X 8 X 9 X 10 (where X 1 is V or I, X 2 is I or V, X 3 is V or I, X 4 is
  • amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 the protein of (a), (d) or (e) An amino acid sequence having an identity of 90% or more with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 4; An amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 6; Amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 8 or the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10 And an amino acid sequence having an identity of 50% or more or 90% or more with the amino acid sequence of the 264 to 423 amino acid residue of the amino acid sequence shown in SEQ ID NO: 2, An amino acid sequence having at least 90% identity with the amino acid sequence of the 264 to
  • amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 is General formula (2): KX 11 PAINIX 12 RTWNAEREX 13 X 14 EX 15 ID (Wherein X 11 is L or I, X 12 is K or Y, X 13 is A or T, X 14 is G or N, and X 15 is K or R) Array, Amino acid sequence having at least 90% identity to the amino acid sequence of amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 or amino acid sequence of amino acid residue 246 to 267 of the amino acid sequence shown in SEQ ID NO: 14 The method according to [12] or [13], wherein the amino acid sequence is 90% or more.
  • the protein according to any one of [7] to [10], the protein produced by the method according to any one of [12] to [17], or the epimerylation according to [21] in galactose A method for producing talose, which comprises causing an agent to act to produce talose.
  • the present invention it is simpler and more efficient by using low cost glucose as a raw material, and epimerizing glucose to mannose respectively by a plurality of MEs found by the present inventors to have new substrate specificity. Mannose can be produced.
  • the pH stability test result of DfME is shown.
  • the heat stability test result of DfME is shown.
  • the reaction test result with respect to the various substrates of Emtol_1243 gene product (EoME) is shown.
  • the reaction test result with respect to various substrates of EoME is shown.
  • the reaction test result with respect to the various substrates of Slin_6381 gene product (SlME) is shown.
  • the reaction test result with respect to various substrates of SlME is shown.
  • the reaction test result with respect to the various substrates of Spirs_3060 gene product (SsME) is shown.
  • the reaction test result with respect to various substrates of SsME is shown.
  • the reaction test result with respect to various substrates of Trebr — 2067 gene product (TbME) is shown.
  • the reaction test result with respect to various substrates of TbME is shown.
  • the reaction test result with respect to various substrates of Treaz_2550 gene product (TaME) is shown.
  • the reaction test result with respect to various substrates of TaME is shown.
  • Amino acid sequences of proteins Runsl_ 4512 and Dfer_ 5652 near the insertion sequence comparison table with primary amino acid sequences of unknown functions and known CE and the like.
  • the primary amino acid sequence comparison chart of the protein of unknown function including the protein Runsl 4512 and near the insertion sequence.
  • the nucleotide sequence of Runsl — 4512 gene (SEQ ID NO: 1) and the amino acid sequence of Runsl — 4512 protein encoded by this nucleotide sequence (SEQ ID NO: 2) are shown.
  • the nucleotide sequence (SEQ ID NO: 3) of the Dfer — 5652 gene and the amino acid sequence (SEQ ID NO: 4) of the Dfer — 5652 protein encoded by this nucleotide sequence are shown.
  • the nucleotide sequence of Emtol_1243 gene (SEQ ID NO: 5) and the amino acid sequence of Emtol_1243 protein encoded by this nucleotide sequence (SEQ ID NO: 6) are shown.
  • the nucleotide sequence (SEQ ID NO: 7) of the Slin — 3681 gene and the amino acid sequence (SEQ ID NO: 8) of the Slin — 3681 protein encoded by this nucleotide sequence are shown.
  • the nucleotide sequence of the Spirs_3060 gene (SEQ ID NO: 9) and the amino acid sequence of the Spirs_3060 protein encoded by this nucleotide sequence (SEQ ID NO: 10) are shown.
  • the nucleotide sequence of Trebr — 2067 gene (SEQ ID NO: 11) and the amino acid sequence of Trebr — 2067 protein encoded by this nucleotide sequence (SEQ ID NO: 12) are shown.
  • the nucleotide sequence of Treaz — 2550 gene (SEQ ID NO: 13) and the amino acid sequence of Treaz — 2550 protein encoded by this nucleotide sequence (SEQ ID NO: 14) are shown.
  • the present invention relates to a protein having an epimerization activity of glucose and mannose having the following amino acid sequence (a) or (b): However, the protein having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14 is excluded.
  • (A) has an insertion sequence of an amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2, An amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 on the N terminal side of the inserted sequence, and SEQ ID NO: 2 on the C terminal side of the inserted sequence
  • (B) has an insertion sequence of an amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12, An amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 at the N-terminal side of the inserted sequence, and SEQ ID NO: 12 at the C-terminal side of the inserted sequence A protein having an amino acid sequence having 50% or more identity to the amino acid sequence of the 263-419 amino acid residue of the amino acid sequence shown in and a glucose and mannose epimerization activity.
  • Runella slithyformis (DSM 19594) has three kinds of genes presumed to encode N-acylglucosamine 2-epimerase (AGE) on the genome ( Gene Bank).
  • AGE N-acylglucosamine 2-epimerase
  • two of the three had CE activity, but the remaining one did not have AGE activity and CE activity.
  • the reactivity to various carbohydrates was examined (Example 1).
  • proteins without this CE activity surprisingly acted on glucose and mannose to produce mannose and glucose, respectively. That is, it was revealed that the protein is mannose 2-epimerase (ME) which mutually converts glucose and mannose.
  • ME mannose 2-epimerase
  • the protein having no CE activity is a protein (Runsl — 4512) having an amino acid sequence of 1 to 423 amino acid residues shown in SEQ ID NO: 2.
  • the base sequence of Runsl 4512 gene is the base sequence of SEQ ID NO: 1, and from this base sequence, Runsl 4512 is published in Gene Bank to be a protein consisting of the amino acid sequence shown in SEQ ID NO: 2 (Gene Bank) ID: WP_013930124).
  • the protein (Runsl — 4512) having the amino acid sequence of 1 to 423 amino acid residues shown in SEQ ID NO: 2 was unknown in function prior to the present invention.
  • a gene encoding a protein presumed to be AGE was further cloned from Dyadobacter fermentans (DSM 18053), and the reactivity of the recombinant enzyme to various carbohydrates was examined (Example) 2). As a result, it was revealed that there is a protein having ME activity (Dfer — 5652) among proteins presumed to be AGE.
  • the nucleotide sequence of the Dfer_ 5652 gene is the nucleotide sequence of SEQ ID NO: 3, and from this nucleotide sequence, the protein (Dfer_ 5652) consists of the amino acid sequence of 1 to 423 amino acid residues shown in SEQ ID NO: 4 to Gene Bank. It has been published (Gene Bank ID: WP_015815082). However, the function of the protein (Dfer — 5652) was unknown before the present invention.
  • Runsl 4512 and Dfer_ 5652 have specific insertion sequences (hereinafter sometimes referred to simply as insertion sequences) in comparison with known primary amino acid sequences such as CE (Fig. 23a-c), It was estimated that the presence of the sequence would be extremely important functionally as ME.
  • This insertion sequence is the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2. Then, a protein of unknown nature is searched for on the database as N-acylglucosamine 2-epimerase (AGE) and the like, and further having a sequence having a certain identity with this insertion sequence, and additionally 5 functions of proteins It was investigated.
  • AGE N-acylglucosamine 2-epimerase
  • Emtisticia oligotrophica Emticicia oligotrophica
  • Spirosoma ringgare Spirosoma linguale
  • Sedimini spilocator Semaradinae Sediminispirochaeta smaragdinae
  • Treponema brennaborens Treponema brennaborense
  • Treponeticium It is a protein encoded by existing genes such as AGE and the like and genes deduced on the database, and the reactivity of these proteins to various carbohydrates was examined.
  • the nucleotide sequences of Emtol_1243, Slin_6381, Spirs_3060, Trebr_2060 and Treaz_2550 genes are the nucleotide sequences of SEQ ID NOs: 5, 7, 9, 11 and 13, respectively, and from this nucleotide sequence, proteins (Emtol_1243, Slin_6381, Spirs_3060, Trebr_2060 and Treaz_2550) has been published to Gene Bank as consisting of amino acid sequences represented by SEQ ID NOs: 6, 8, 10, 12 and 14, respectively (Gene Bank ID: WP — 0150280292, ADB 42339, WP — 013255620, WP — 013759185 and WP — 015712977, respectively). However, these proteins were unknown in function prior to the present invention.
  • amino acid sequence having 70% or more identity with the amino acid sequence of the 241 to 263 amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 in the protein of (a) preferably has 80% or more, 90% or more identity , 95% or more, 98% or more, or 99% or more.
  • an amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2 in the protein of (a) is, for example, X 1 APQIKFDX 2 X 3 WGWDRFX 4 X 5 X 6 X 7 X 8 X 9 X 10 (where X 1 is V or I, X 2 is I or V, X 3 is V or I, X 4 is N, T or S, X 5 is P or E, X 6 is D or G, X 7 is G or D, X 8 is L, V, Q or A , X 9 is K or Q and X 10 is S, A, E or K).
  • the amino acid sequence of the general formula (1) is based on the insertion sequence in the amino acid sequence represented by SEQ ID NO: 2, 4, 6, 8 and 10, and X 1 , X 2 , X 3 , X 4 , X 5 , X 6 , X 7 , X 8 , X 9 and X 10 are those determined from variations in these insertion sequences.
  • the amino acid sequence having 70% or more identity with the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2 in the protein of (a) is, for example, 241 of the amino acid sequence shown in SEQ ID NO: 2 An amino acid sequence having at least 90% identity with the amino acid sequence of -263 amino acid residues, An amino acid sequence having at least 90% identity to the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 4; An amino acid sequence having at least 90% identity with the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 6; Amino acid sequence having at least 90% identity to the amino acid sequence of amino acid sequence 241 to 263 of the amino acid sequence shown in SEQ ID NO: 8 or amino acid sequence of amino acid residue 241 to 264 of the amino acid sequence shown in SEQ ID NO: 10 And an amino acid sequence having an identity of 90% or more.
  • amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 in the protein of (a) preferably has 60% or more, 70% or more identity 80% or more, 90% or more, or 95% or more.
  • an amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 in the protein of (a) is, for example, An amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2; An amino acid sequence having an identity of 90% or more with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 4; An amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 6; Amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 8 or the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10 And an amino acid sequence having an identity of 90% or more.
  • amino acid sequence having 50% or more identity with the amino acid sequence of the 264 to 423 amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 in the protein of (a) preferably has 60% or more, 70% or more identity 80% or more, 90% or more, or 95% or more.
  • an amino acid sequence having 50% or more identity to the amino acid sequence of the 264 to 423 amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 in the protein of (a) is, for example, An amino acid sequence having at least 90% identity to the amino acid sequence of the 264 to 423 amino acid residue of the amino acid sequence shown in SEQ ID NO: 2; An amino acid sequence having at least 90% identity with the amino acid sequence of the 264 to 423 amino acid residue of the amino acid sequence shown in SEQ ID NO: 4; An amino acid sequence having at least 90% identity with the amino acid sequence of the 264 to 422 amino acid residues of the amino acid sequence shown in SEQ ID NO: 6; Amino acid sequence having at least 90% identity with the amino acid sequence of the 264 to 427 amino acid residue of the amino acid sequence shown in SEQ ID NO: 8 or the amino acid sequence of the 265 to 428 amino acid residue of the amino acid sequence shown in SEQ ID NO: 10 And an amino acid sequence having an identity of 90% or more.
  • amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (b) preferably has an identity of 80% to 90%. , 95% or more, 98% or more, or 99% or more.
  • an amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (b) is, for example, KX 11 PAINIX 12 RTWNAEREX 13 X 14 EX 15 ID (Wherein X 11 is L or I, X 12 is K or Y, X 13 is A or T, X 14 is G or N, and X 15 is K or R) It can be an array.
  • the amino acid sequence of the general formula (2), based on the insertion sequence in the amino acid sequence represented by SEQ ID NO: 12 and 14, and X 11, X 12, X 13 , X 14, X 15 is variations in these insertion sequences It is determined from
  • the amino acid sequence having 70% or more identity with the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (b) is the amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12.
  • amino acid sequence having 50% or more identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (b) preferably has 60% or more, 70% or more identity 80% or more, 90% or more, or 95% or more.
  • an amino acid sequence having 50% or more identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (b) is Amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues, or amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 245 amino acid residues of the amino acid sequence shown in SEQ ID NO: 14 Can be.
  • amino acid sequence having 50% or more identity with the amino acid sequence of the 263-419 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (b) preferably has 60% or more, 70% or more identity 80% or more, 90% or more, or 95% or more.
  • an amino acid sequence having 50% or more identity with the amino acid sequence of the 263-419 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (b) is, for example, the amino acid sequence shown in SEQ ID NO: 12
  • Amino acid sequence having at least 90% identity with the amino acid sequence of 263-419 amino acid residues, or at least 90% identity with the amino acid sequence of 268-430 amino acid residues of the amino acid sequence shown in SEQ ID NO: 14 Can be.
  • an amino acid sequence having 90% or more identity with the amino acid sequence is preferably an amino acid sequence having 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identity. is there.
  • the fact that the protein is a protein having an epimerization activity of glucose and mannose can be confirmed by the following activity measurement method described also in the examples.
  • Activity measurement Enzyme reaction was carried out by holding 100 ⁇ L of a reaction solution containing 10 mM mannose, 40 mM HEPES-NaOH buffer (pH 8.0), and protein (enzyme) at 37 ° C for 20 minutes and heating at 80 ° C for 5 minutes Stop. The amount of glucose contained in 50 ⁇ L of the supernatant obtained by centrifugation at 12000 rpm for 10 minutes is quantified by the hexokinase method using Thio-NAD + .
  • the hexokinase method is 0.5 mM Thio-NAD + , 7 mM ATP, 0.1 M Tris-HCl (pH 7.5), 1.0 mM MgCl 2 , 1.0 U / mL hexokinase (from baker's yeast, nacalai tesque) and 1.0 U / mL G6P
  • An equal amount of a coloring reagent containing dehydrogenase (Nacalai Tesque) is added to the reaction solution and kept at 37 ° C. for 20 minutes, and the absorbance of Thio-NADH generated along with the oxidation reaction of G6P is a spectrophotometer with a spectrophotometer at 405 nm.
  • Measure by Enzyme activity 1 U is defined as the amount of enzyme that produces 1 ⁇ mol glucose per minute.
  • the proteins having epimerization activity of glucose and mannose according to the present invention include proteins further having epimerization activity of galactose and talose.
  • the protein further having epimerizing activity of galactose and talose can be confirmed by the following activity measurement method described also in the examples.
  • Activity measurement Enzyme reaction is carried out by holding 100 ⁇ L of a reaction solution containing 10 mM galactose, 40 mM HEPES-Na buffer (pH 8.0), and protein (enzyme) at 37 ° C for 20 minutes and heating at 80 ° C for 5 minutes Stop.
  • the reaction stop solution is subjected to HPLC analysis to detect talose produced by the enzyme reaction.
  • HPLC is performed using a “HILICpak VG-504E” column (manufactured by Shodex) at a column temperature of 40 ° C., a flow rate of 0.6 ml / min, and a solvent of 80% acetonitrile.
  • Enzyme activity 1 U is defined as the amount of enzyme that produces 1 ⁇ mol talose per minute.
  • the present invention includes glucose and mannose epimerizing agents containing the above-mentioned protein of the present invention as an active ingredient.
  • the epimerization agent only needs to be capable of epimerizing glucose and mannose, and may contain materials used for ordinary enzyme preparations such as other enzymes, stabilizers, excipients, etc. in addition to the above-mentioned protein of the present invention Good.
  • the shape is also not particularly limited, and may be powder or liquid, or may be immobilized enzyme immobilized on a carrier.
  • the epimerising agent of the present invention can be used, for example, by adding it to an aqueous solution of glucose which is a substrate.
  • the present invention relates to a protein having the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14 or to the amino acid sequence of the protein It includes an epimerising agent for glucose and mannose, which has an amino acid sequence of 70% or more and a protein having an epimerizing activity for glucose and mannose as an active ingredient.
  • the proteins having the amino acid sequences shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14 are Runsl_ 4512, Dfer_ 5652, Emtol_1243, Slin_6381, Spirs_3060, Trebr_2067 respectively. And Treaz_2550 proteins, which, as described above, have ME activity.
  • the epimerising agent of the present invention has 70% or more identity with the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14. %, Preferably 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more of the amino acid sequences, and have an epimerylation activity of glucose and mannose It contains protein as an active ingredient.
  • the protein having epimerization activity of glucose and mannose can be confirmed by the above-mentioned activity measurement method.
  • the present invention also encompasses a protein having an epimerization activity of galactose and talose having the following amino acid sequence (c) or (d): However, the amino acid sequence shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14 from the protein having epimerization activity of galactose and talose according to the present invention
  • To remove proteins that have (C) has an insertion sequence of an amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 264 of the amino acid sequence shown in SEQ ID NO: 10, An amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10 at the N-terminal side of the inserted sequence, and SEQ ID NO: 10 at the C-terminal side
  • (D) has an insertion sequence of an amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12, An amino acid sequence having 50% or more identity to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 at the N-terminal side of the inserted sequence, and SEQ ID NO: 12 at the C-terminal side of the inserted sequence A protein having an amino acid sequence of 50% or more identity to the amino acid sequence of the 263-419 amino acid residue of the amino acid sequence shown in and a protein having an epimerylation activity of galactose and talose.
  • the protein having the amino acid sequence (1-428 amino acid residues) shown in SEQ ID NO: 10 in the protein of (c) is a Spirs_3060 protein, which has, in addition to the glucose and mannose epimerization activities, galactose and It also has epimerization activity of Tulose.
  • the amino acid sequence having 70% or more identity with the amino acid sequence of the 241 to 264 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10 in the protein of (c) preferably has 80% or more, 90% or more identity , 95% or more, 98% or more, or 99% or more. Furthermore, an amino acid sequence having 70% or more identity with the amino acid sequence of the 241 to 264 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10 in the protein of (c) is the 241 of the amino acid sequence shown in SEQ ID NO: 10
  • the amino acid sequence may have 90% or more identity with the amino acid sequence of -264 amino acid residues.
  • the amino acid sequence having 50% or more identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10 in the protein of (c) preferably has 60% or more, 70% or more identity 80% or more, 90% or more, or 95% or more. Furthermore, an amino acid sequence having 50% or more identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10 in the protein of (c) is The amino acid sequence may have an identity of 90% or more with the amino acid sequence of 1 to 240 amino acid residues.
  • the amino acid sequence having 50% or more identity with the amino acid sequence of the 265 to 428 amino acid residue of the amino acid sequence shown in SEQ ID NO: 10 in the protein of (c) preferably has 60% or more, 70% or more identity 80% or more, 90% or more, or 95% or more. Furthermore, the amino acid sequence having 50% or more identity to the amino acid sequence of the 265 to 428 amino acid residue of the amino acid sequence shown in SEQ ID NO: 10 in the protein of (c) is the amino acid sequence shown in SEQ ID NO: 10
  • the amino acid sequence may have an identity of 90% or more with the amino acid sequence of 428 amino acid residues.
  • amino acid sequence having 70% or more identity with the amino acid sequence of the 241 to 262 amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (d) preferably has 80% or more, 90% or more identity , 95% or more, 98% or more, or 99% or more.
  • an amino acid sequence having 70% or more identity with the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (d) has a general formula (2): KX 11 PAINIX 12 RTWNAEREX 13 X 14 EX 15 ID (Wherein X 11 is L or I, X 12 is K or Y, X 13 is A or T, X 14 is G or N, and X 15 is K or R) Array, Amino acid sequence having at least 90% identity to the amino acid sequence of amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 or amino acid sequence of amino acid residue 246 to 267 of the amino acid sequence shown in SEQ ID NO: 14 And an amino acid sequence having an identity of 90% or more.
  • the protein having the amino acid sequence (1-419 amino acid residues) shown in SEQ ID NO: 12 in the protein of (d) is Trebr — 2067 protein, and the amino acid sequence (1-34 amino acid residues) shown in SEQ ID NO: 14
  • the protein having is Treaz_2550 protein, and these proteins also have the epimerization activity of galactose and talose in addition to the epimerization activity of glucose and mannose.
  • amino acid sequence having 50% or more identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (d) preferably has 60% or more, 70% or more identity 80% or more, 90% or more, or 95% or more.
  • an amino acid sequence having 50% or more identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (d) is, for example, Amino acid sequence having 90% or more identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 or with amino acid sequence 1 to 245 amino acid residue of the amino acid sequence shown in SEQ ID NO: 14
  • the amino acid sequence may be 90% or more in sex.
  • the amino acid sequence having 50% or more identity with the amino acid sequence of the 263-419 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (d) preferably has 60% or more, 70% or more identity 80% or more, 90% or more, or 95% or more.
  • an amino acid sequence having 50% or more identity to the amino acid sequence of the 263-419 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (d) is, for example, the amino acid sequence shown in SEQ ID NO: 12
  • Amino acid sequence having at least 90% identity with the amino acid sequence of 263 to 419 amino acid residue of the amino acid sequence or amino acid with at least 90% identity with the amino acid sequence of It can be an array.
  • the origin of the protein of the present invention is not particularly limited.
  • the protein of the present invention may be a recombinant protein produced by various genetic engineering techniques, or a synthetic protein produced by chemical synthesis.
  • nucleic acid DNA or RNA
  • a nucleic acid (DNA or RNA) encoding the above-mentioned protein of the present invention is prepared and incorporated into various expression vectors to express the protein of the present invention It can be done.
  • preparing the nucleic acid encoding the protein of the present invention for example, error-prone PCR, DNA shuffling, various site-directed mutagenesis, etc. can be used to carry out deletion, substitution and / or addition of amino acids. Deletion, substitution and / or insertion of any base can be performed.
  • the protein of the present invention can be produced by introducing the thus prepared nucleic acid encoding the protein of the present invention into an appropriate expression system.
  • the expression system that can be used to produce the protein of the present invention is not particularly limited, and for example, expression vectors that allow expression of recombinant proteins in various species (hosts) can be used.
  • expression vectors that allow expression of proteins in hosts such as microorganisms such as bacteria and fungi (for example, yeasts), plants, insect cells, and mammalian cells, It may be a viral vector (including a phage vector) or a plasmid vector.
  • the protein of the present invention may be produced using a cell-free protein expression system using rabbit reticulocyte lysate, wheat germ lysate, E. coli lysate or the like.
  • the nucleic acid encoding the above-mentioned protein of the present invention is loaded on a vector, and the host cell is transformed with this vector,
  • the transformed host cells can be cultured, and the protein encoded by the gene can be accumulated in the culture, and the accumulated protein can be prepared by a production method.
  • nucleic acid encoding the protein of the present invention a vector containing the nucleic acid, and a transformant transformed with the vector are one aspect of the present invention.
  • the method for obtaining the nucleic acid encoding the protein of the present invention is not particularly limited.
  • a nucleic acid encoding the protein of the present invention may be used as a material to obtain a nucleic acid encoding the protein of the present invention, or a nucleic acid encoding the protein of the present invention may be prepared Then, appropriate substitutions and the like may be carried out to produce a nucleic acid encoding the protein of the present invention.
  • the nucleic acid encoding the protein of the present invention can be produced by any method known to those skilled in the art, such as chemical synthesis, genetic engineering or mutagenesis.
  • the protein of the present invention can be appropriately prepared by the existing method if the amino acid sequence or the nucleotide sequence encoding it is known.
  • the present invention also includes the following production methods (1) and (2).
  • the method (1) for producing a protein having epimerization activity of glucose and mannose and / or epimerization activity of galactose and talose according to the present invention has epimerization activity of glucose and mannose and epimerization activity of galactose and talose From unknown proteins, (a) a protein having an amino acid sequence of 70% or more identity to the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2, or (b) SEQ ID NO: 12 Selecting a protein having an amino acid sequence having an identity of 70% or more with the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown in (c), and from the proteins selected in (c) (a) or (b), Glucose and mannose epimerization activity, galactose Comprising selecting a protein having an epimerization activity or both talose and.
  • the selection of the protein of (a) or (b) above can be carried out based on the amino acid sequence information of the protein whose amino acid sequence is known.
  • a protein having an insertion sequence shown in the following Table 5 and FIGS. 23b and 23c can be exemplified.
  • selection of a protein having an epimerylation activity of glucose and mannose, an epimerylation activity of galactose and talose, or both of them prepares a DNA having a nucleotide sequence encoding an amino acid sequence of the selected protein It can be expressed by expressing it and measuring the enzyme activity. DNA preparation and expression can be performed by the aforementioned existing methods. The enzyme activity measurement is as described above.
  • the method (2) for producing a protein having epimerization activity of glucose and mannose and / or epimerization activity of galactose and talose comprises (d) the amino acid sequence of the known or unknown amino acid sequence of SEQ ID NO: 2 An amino acid sequence identical to that of amino acid sequence 241 to 263 of the amino acid sequence shown in the amino acid sequence 70% or more or an amino acid sequence identical to that of amino acid residue 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 % Or more of the amino acid sequence, or (e) amino acid sequence of at least a portion of the amino acid sequence of the known or unknown protein, amino acid of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2 Amino acid sequence of 70% or more identity to the sequence or shown in SEQ ID NO: 12 And (f) from the protein inserted or modified in (d) or (e), an amino acid sequence having an identity of 70% or more with the amino acid sequence of 241 to 262 amino acid residues of the amino
  • sequence insertion and modification to the above-mentioned (d) or (e) protein can be carried out based on the amino acid sequence information of the protein whose amino acid sequence is known or unknown.
  • the protein shown in FIG. 23a can be mentioned.
  • selection of a protein having an epimerylation activity of glucose and mannose, an epimerylation activity of galactose and talose, or both prepares a DNA having a nucleotide sequence encoding the amino acid sequence of the selected protein, This can be expressed by expressing it and measuring the enzyme activity.
  • sequence insertion and modification to the protein of (d) or (e) above can be preferably performed on an amino acid sequence having an amino acid sequence identity of 70% or more with CE described in Patent Documents 3 and 4.
  • sequence insertion and modification to the protein of (d) or (e) above can also be performed on a protein of which the amino acid sequence is shown in FIG.
  • the protein of which the amino acid sequence is known has a known function. Even if the function is unknown, it may be an amino acid sequence having no insertion sequence specified in the present invention.
  • the insertion sequence specified in the present invention can be inserted, and if necessary, modification of the insertion sequence part or the sequences before and after it can be performed.
  • insertion of the insertion sequence specified in the present invention into a protein of known function it may be modified into a protein having epimerization activity of glucose and mannose, epimerization activity of galactose and talose, or both.
  • DNA preparation and expression can be performed by the aforementioned existing methods. The enzyme activity measurement is as described above.
  • the amino acid sequence having 70% or more identity with the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2 in (a), (d) or (e) preferably has 80% identity.
  • the protein prepared in (c) or (f) through (a), (d) or (e) is identical to the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2 Amino acid sequence of 50% or more or 90% or more, An amino acid sequence having an identity of 90% or more with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 4; An amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 6; Amino acid sequence having at least 90% identity with the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 8 or the amino acid sequence of 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 10 And an amino acid sequence having an identity of 50% or more or 90% or more with the amino acid sequence of the 264 to 423 amino acid residue of the
  • the amino acid sequence having 70% or more identity with the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 in (b), (d) or (e) preferably has 80% identity %, 90% or more, 95% or more, 98% or more, or 99% or more, for example, General formula (2): KX 11 PAINIX 12 RTWNAEREX 13 X 14 EX 15 ID (Wherein X 11 is L or I, X 12 is K or Y, X 13 is A or T, X 14 is G or N, and X 15 is K or R) Array, Amino acid sequence having at least 90% identity to the amino acid sequence of amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 or amino acid sequence of amino acid residue 246 to 267 of the amino acid sequence shown in SEQ ID NO: 14 And an amino acid sequence having an identity of 90% or more.
  • the protein prepared in (c) or (f) via (b), (d) or (e) is a protein of (b) having 1 to 240 amino acid residues of the amino acid sequence shown in SEQ ID NO: 12 Amino acid sequence having at least 50% or at least 90% identity with the amino acid sequence of the group, or at least 90% with the amino acid sequence of 1 to 245 amino acid residues of the amino acid sequence shown in SEQ ID NO: 14 Furthermore, an amino acid sequence having 50% or more or 90% or more identity with the amino acid sequence of the 263-419 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence shown in SEQ ID NO: 14
  • the protein may further have an amino acid sequence of 90% or more identity to the amino acid sequence of 268 to 430 amino acid residues.
  • the production methods (1) and (2) of the present invention are production of a protein having epimerization activity of glucose and mannose.
  • the production method of the present invention can also be production of a protein having epimerization activity of galactose and talose.
  • measurement of enzyme activity may be performed for the epimerization activity of galactose and talose.
  • the present invention includes a method for producing mannose, and the method for producing mannose according to the present invention comprises, in glucose, the protein having epimerization activity of glucose and mannose according to the present invention, the epimerising agent according to the present invention or the method according to the present invention Allowing the produced protein to act to produce mannose.
  • the production of mannose from glucose using the protein etc. of the present invention can be carried out, for example, by preparing an aqueous solution of carbohydrate having a solid content concentration of about 1 to 50% containing glucose, and using pH of about 6 to 10 using acid or alkali as necessary.
  • Mannose can be produced by adding the protein of the present invention to the solution and maintaining the solution at 30 to 60.degree. C. for 1 to 72 hours. After the above enzyme reaction, the mannose-containing carbohydrate composition can be subjected to decolorization, desalting, purification treatment and the like by using a conventional method as necessary. Furthermore, by subjecting the obtained mannose-containing carbohydrate composition to a resin fractionation treatment or the like, mannose and glucose as a substrate can be separated to increase the mannose purity. Alternatively, mannose (a mannose-containing carbohydrate composition) can be produced by immobilizing the protein of the present invention on a carrier to form an immobilized enzyme, and bringing the immobilized enzyme into contact with the substrate solution.
  • a sugar composition containing other carbohydrates may be used from the viewpoint of cost etc.
  • starch syrup powdered starch
  • hydrol liquid separation produced during crystalline glucose production
  • a sugar containing glucose as a constituent sugar such as dextrin and starch is used as a raw material, and a hydrolase is added together with the protein of the present invention to produce mannose from glucose while producing glucose as a substrate from the raw material. it can.
  • the present invention encompasses epimerising agents for galactose and talose.
  • the epimerising agent for galactose and talose according to the present invention has a protein having the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14 or an amino acid sequence of 70% or more and 75% or more
  • a protein having an epimerization activity of galactose and talose with an identity of 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more is used as an active ingredient.
  • the present invention includes a method for producing talose, the method for producing talose according to the present invention comprising galactose, the protein having epimerizing activity of galactose and talose according to the present invention, or galactose and talose produced according to the method of the present invention
  • a protein having an epimerization activity, or galactose of the present invention and an epimerizing agent of talose are reacted to produce talose.
  • talose talose-containing carbohydrate composition
  • the talose-containing carbohydrate composition can be subjected to a decolorization, desalting, purification treatment and the like using a conventional method.
  • talose a talose-containing carbohydrate composition
  • talose a talose-containing carbohydrate composition
  • talose can also be produced by immobilizing the protein of the present invention on a carrier to obtain an immobilized enzyme, and bringing the immobilized enzyme into contact with the substrate solution.
  • a pure galactose product may be used as a substrate for producing talose from galactose, but a sugar composition containing other carbohydrates may be used from the viewpoint of cost and the like.
  • a saccharide containing galactose such as lactose as a constituting sugar is used as a raw material, and a hydrolase is added together with the protein of the present invention, and talose can be produced from galactose while producing galactose as a substrate from the raw material.
  • Example 1 In this example, a gene encoding a protein of unknown function (Runsl — 4512), which is presumed to be N-acylglucosamine 2-epimerase (AGE) from Runella slithyformis, was cloned, and the recombinant enzyme was characterized.
  • Unsl — 4512 which is presumed to be N-acylglucosamine 2-epimerase (AGE) from Runella slithyformis
  • Runsl 4512 gene was cloned from Slithyformis (DSM19594).
  • the base sequence of Runsl 4512 gene is the base sequence of SEQ ID NO: 1, and from the base sequence, Runsl — 4512 is published in Gene Bank as a protein consisting of the amino acid sequence shown in SEQ ID NO: 2.
  • Runsl — 4512 Gene Product The Runsl — 4512 gene shown in SEQ ID NO: 1 from which the stop codon was removed was introduced into the Nde1-Xho1 site of pET23a to construct a Runsl_ 4512 expression vector to which a histidine tag was added.
  • the expression vector was introduced into E. coli BL21 (DE3) to prepare a transformant.
  • the transformant was inoculated into 30 mL of LBA medium containing 100 ⁇ g / mL of ampicillin and cultured overnight at 37 ° C. This was placed in 1 L of LBA medium, and cultured at 37 ° C. until the absorbance (A 600 ) at a wavelength of 600 nm became 0.5.
  • the obtained cell-free extract was subjected to nickel ion chelated Chelating Sepharose Fast Flow (GE Healthcare) to purify recombinant Runsl 4512. That is, after washing the carrier with 30 mmol / L imidazole-HCl buffer (pH 7) containing 0.5 mol / L NaCl, elution with 250 mmol / L imidazole-HCl buffer (pH 7) containing 0.5 mol / L NaCl did. The obtained sample was dialyzed into 10 mmol / L Tris-HCl buffer (pH 7) and used for further analysis. The samples were analyzed by SDS-PAGE to confirm a single band.
  • the enzyme reaction was terminated by holding the reaction solution containing 10 mM mannose, 40 mM Tris-HCl buffer (pH 7.8) and 0.1 mL of the reaction solution at 37 ° C. for 10 minutes and heating at 80 ° C. for 5 minutes.
  • the amount of glucose contained in 50 ⁇ L of the supernatant obtained by centrifugation at 12000 rpm for 5 minutes was quantified by the hexokinase method using an F-kit.
  • the specific activity of Runsl 4512 obtained under the reaction conditions was 0.864 U / mg.
  • the enzyme activity 1 U was defined as the amount of enzyme that produces 1 ⁇ mol glucose per minute.
  • Optimal pH The enzyme reaction was terminated by holding 0.1 mL of a reaction solution containing 10 mM mannose, 80 mM Britton-Robinson buffer (pH 6-10) and enzyme at 37 ° C. for 10 minutes and heating at 80 ° C. for 5 minutes. Glucose production was determined as described above. The results are shown in FIG. The optimum pH was 7.8.
  • pH stability A 0.1 mL solution consisting of the enzyme and 0.1 M Britton-Robinson buffer (pH 3-12) was kept at 4 ° C. for 24 hours. The enzyme activity was measured as described above. The results are shown in FIG. It was stable in the range of pH 6.3-9.8.
  • Temperature Stability 0.1 mL of the enzyme solution was kept at 30 to 60 ° C. for 15 minutes, and this activity was measured as described above. The results are shown in FIG. It was stable below 40 ° C.
  • Example 2 a gene encoding a protein (Dfer — 5652) presumed to be N-acylglucosamine 2-epimerase (AGE) was cloned from Diadobacter fermentans, and the recombinant enzyme was characterized.
  • Dfer — 5652 presumed to be N-acylglucosamine 2-epimerase
  • Dfer_5652 gene D Cloning of Dfer_5652 gene D.
  • the Dfer — 5652 gene was cloned from F. fermentans (DSM 18053).
  • the nucleotide sequence of the Dfer — 5652 gene is disclosed in Gene Bank as being the nucleotide sequence of SEQ ID NO: 3, and from the nucleotide sequence, Dfer — 5652 is a protein consisting of the amino acid sequence shown in SEQ ID NO: 4.
  • Dfer — 5652 gene shown in SEQ ID NO: 3 from which the termination codon had been removed was introduced into the Nde1-Xho1 site of pET23a to construct a Dfer_5652 expression vector to which a histidine tag was added.
  • the expression vector was introduced into E. coli BL21 (DE3) to prepare a transformant.
  • the transformant was inoculated into 30 mL of LBA medium containing 100 ⁇ g / mL of ampicillin and cultured overnight at 37 ° C. This was placed in 1 L of LBA medium, and cultured at 37 ° C. until the absorbance (A 600 ) at a wavelength of 600 nm became 0.5.
  • the culture solution was ice-cooled, and 1 mL of 0.1 mol / L isopropyl ⁇ -thiogalactopyranoside was added to a final concentration of 0.1 mmol / L, followed by culturing at 37 ° C. for 4 hours.
  • the culture solution was centrifuged at 7,000 ⁇ g at 4 ° C. for 10 minutes to recover the cells.
  • the cells were suspended in 30 mmol / L imidazole-HCl buffer (pH 7) containing 0.5 mol / L NaCl, and the cells were disrupted by sonication.
  • the resulting disrupted cell suspension was centrifuged at 10,000 ⁇ g at 4 ° C. for 10 minutes, and the supernatant was recovered.
  • the resulting cell-free extract was subjected to nickel ion chelated Chelating Sepharose Fast Flow (GE Healthcare) to purify recombinant Dfer — 5652. That is, after washing the carrier with 30 mmol / L imidazole-HCl buffer (pH 7) containing 0.5 mol / L NaCl, elution with 500 mmol / L imidazole-HCl buffer (pH 7) containing 0.5 mol / L NaCl did. The obtained sample was dialyzed into 10 mmol / L Tris-HCl buffer (pH 7) and used for further analysis. The samples were analyzed by SDS-PAGE to confirm a single band.
  • TLC analysis of the reaction product 5 ⁇ L of a reaction solution consisting of 50 mM various substrates, 5 mM Tris-HCl (pH 8.0) and 0.0410 mg / ml Dfer — 5652 was kept at 37 ° C. overnight, and 1 ⁇ L of this was subjected to TLC.
  • the developing solvent is shown below.
  • the developing solvent used was different for each substrate.
  • Optimal pH The enzyme reaction was terminated by holding 100 ⁇ L of a reaction solution containing 10 mM mannose, 40 mM Britton Robinson buffer (pH 5.5-10.5) and the enzyme at 37 ° C. for 20 minutes and heating at 80 ° C. for 5 minutes. Glucose production was determined as described above. The results are shown in FIG. The optimum pH was 8.1.
  • pH stability 100 ⁇ L of a solution consisting of enzyme and 50 mM Britton-Robinson buffer (pH 5.5-12.5) was kept at 4 ° C. for 24 hours. This solution was used as an enzyme solution to measure the activity in the same manner as described above. The results are shown in FIG. It was stable in the range of pH 6.3-9.6.
  • Temperature stability 60 ⁇ L of the enzyme solution was kept at 20-55 ° C. for 20 minutes, and the activity was measured by the same procedure as the above activity measurement. The results are shown in FIG. The activity of 80% or more was maintained at 45 ° C. or less.
  • Emtitissia oligotrophica Emticicia oligotrophica
  • Spirosoma ringgare Spirosoma linguale
  • Sedimini spilocator Smarajinae Sedimini priochaeta smaragdinae
  • Treponema brenabolens Teponema brennaborense
  • Genes estimated on the database with AGE etc. each encoding Emtol_1243, Slin_6381, Spirs_3060, Trebr_2067 and Treaz_2550, respectively
  • Target Gene was purchased from Eurofin Genomics Ltd. using artificial gene synthesis.
  • the target gene was designed on the company's HP and optimized for the codon usage of the E. coli K12 strain.
  • the synthesized gene was delivered in the state of being inserted into pEX-A2J1 (pUC system, Amp resistance) containing an ampicillin resistance gene.
  • the target gene was inserted into pET23a using In-Fusion HD Cloning Kit (Takara Bio). E. coli DH5 ⁇ was used for plasmid amplification after the In-Fusion reaction. The obtained plasmid was sequence analyzed to confirm that the target gene was correctly inserted.
  • E. coli BL21 E. coli BL21 (DE3).
  • the transformant was cultured overnight at 37 ° C. in 30 mL of LB medium containing 100 mg / L ampicillin, and transferred to 1 L of the same medium.
  • A600 reached 0.5-0.8
  • 1 mL of 0.1 M IPTG was added, and the culture was performed at 18 ° C. for 24 hours.
  • the cells were centrifuged to recover the cells and stored at -30.degree.
  • the cryopreserved pellet was suspended in 30 mM imidazole-HCl buffer (pH 7) containing 0.5 M NaCl.
  • the cells were disrupted by sonication.
  • the supernatant obtained by centrifugation was filtered through a 0.45 ⁇ m filter to obtain a cell-free extract, and the precipitate was suspended in the same amount of the above buffer as the cell-free extract.
  • the cell-free extract was subjected to nickel ion chelated Chelating Sepharose Fast Flow (GE Healthcare) to purify the recombinant enzyme.
  • Activity measurement Hold 100 ⁇ L of reaction solution containing 10 mM mannose, 40 mM HEPES-NaOH buffer (pH 8.0), and enzyme for 30 minutes or 60 minutes at 37 ° C and heat the enzyme reaction by heating at 80 ° C for 5 minutes It stopped.
  • the amount of glucose contained in 50 ⁇ L of the supernatant obtained by centrifugation at 12000 rpm for 10 minutes was quantified by the hexokinase method using Thio-NAD + .
  • the substrate specificity of each enzyme is summarized in Table 2. Since all enzymes were confirmed to produce mannose from glucose and glucose from mannose, it was determined to be mannose 2-epimerase, which catalyzes the interconversion of glucose and mannose. In addition, it was also found that three enzymes of Spirs_3060, Trebr_2067 and Treaz_2550 have a catalytic action to mutually convert galactose and talose. Furthermore, Treaz_2550 also had CE activity and the like.
  • the enzyme of the present invention is presumed to be similar in three-dimensional structure to cellobiose 2-epimerase having an ⁇ / ⁇ -6 barrel structure.
  • a unique sequence common to the enzymes of the present invention, as in CE, constitutes a part of the loop between the ⁇ 7 helix and the ⁇ 8 helix, and the loop is extended as compared to CE.
  • the loop is considered to constitute a part of the active site, and the presence of a sequence unique to this enzyme in the loop changes the substrate recognition mechanism as compared to CE and is preferable for the reaction to glucose and mannose It is assumed that the shape of the active site is obtained.
  • the present invention is useful in the field related to proteins having epimerization activity such as proteins having epimerization activity of glucose and mannose and proteins having epimerization activity of galactose and talose.
  • SEQ ID NO: 1 Runsl — 4512 base sequence
  • SEQ ID NO: 2 Runsl — 4512 amino acid sequence
  • SEQ ID NO: 3 Dfer — 5652 base sequence
  • SEQ ID NO: 4 Dfer — 5652 amino acid sequence
  • SEQ ID NO: 5 Emtol_1243 base sequence
  • SEQ ID NO: 6 Emtol_1243 amino acid sequence
  • SEQ ID NO: 7 Slin_6381 base sequence No.
  • SEQ ID NO: 9 Spirs_3060 base sequence
  • SEQ ID NO: 10 Spirs_3060 amino acid sequence
  • SEQ ID NO: 11 Trebr_2067 base sequence
  • SEQ ID NO: 12 Trebr_2067 amino acid sequence
  • SEQ ID NO: 13 Treaz_2550 base sequence
  • SEQ ID NO: 14 Treaz_2550 amino acid sequence
  • SEQ ID NO: 15 to 46 amino acid sequences of the proteins listed in Tables 6 and 7 7: amino acid sequence of the protein described in Table 5

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Abstract

La présente invention concerne : une protéine comprenant une séquence d'insertion spécifique donnée et affichant une activité d'épimérisation de glucose-mannose, et un procédé de production de mannose à l'aide de ladite protéine ; une protéine comprenant une séquence d'insertion spécifique donnée et affichant une activité d'épimérisation de galactose-talose, et un procédé de production de talose à l'aide de ladite protéine ; des procédés de production des protéines ; et une préparation enzymatique contenant les protéines. La présente invention concerne un procédé permettant de produire respectivement du mannose et du talose efficacement, et une enzyme ou une préparation enzymatique qui peut être utilisée dans le procédé.
PCT/JP2018/030497 2017-08-17 2018-08-17 Protéine présentant une activité d'épimérisation Ceased WO2019035482A1 (fr)

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JP7592539B2 (ja) 2021-04-01 2024-12-02 日本食品化工株式会社 糖のエピメリ化反応触媒用の酵素剤、エピメリ化反応生成物の製造方法およびエピメリ化反応生成物
CN119331799A (zh) * 2024-12-20 2025-01-21 山东百龙创园生物科技股份有限公司 重组枯草芽孢杆菌blcy-013及其在以葡萄糖为原料制备甘露糖中的应用

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JP6959411B1 (ja) 2020-07-31 2021-11-02 日本食品化工株式会社 糖のエピメリ化反応触媒用の酵素剤、エピメリ化反応生成物の製造方法およびエピメリ化反応生成物

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP7592539B2 (ja) 2021-04-01 2024-12-02 日本食品化工株式会社 糖のエピメリ化反応触媒用の酵素剤、エピメリ化反応生成物の製造方法およびエピメリ化反応生成物
CN119331799A (zh) * 2024-12-20 2025-01-21 山东百龙创园生物科技股份有限公司 重组枯草芽孢杆菌blcy-013及其在以葡萄糖为原料制备甘露糖中的应用
CN119331799B (zh) * 2024-12-20 2025-03-04 山东百龙创园生物科技股份有限公司 重组枯草芽孢杆菌blcy-013及其在以葡萄糖为原料制备甘露糖中的应用

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