JP2019033702A - Protein having epimerization activity - Google Patents

Abstract

To provide methods that can efficiently produce mannose and talose respectively, and to provide enzymes or enzyme agents which can be used in the methods.SOLUTION: Provided are a protein having epimerization activity of glucose and mannose and comprising a particular specific insertion sequence, and a method for producing mannose using this protein. Provided are a protein having epimerization activity of galactose and talose and comprising a particular specific insertion sequence, and a method for producing talose using this protein. Methods for producing these proteins are provided. Enzyme agents comprising these proteins are provided.SELECTED DRAWING: None

Description

  The present invention relates to a protein having epimerization activity.

  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.

  The bioavailability of mannose is low and most of the orally ingested mannose is rapidly excreted in the urine. The mechanism that causes the bacteria causing urinary tract infections to attach to and infect urinary tract surface cells through binding of mannose binding protein (lectin) possessed by the bacteria and sugar chains present on urinary tract surface cells is proposed It is done. As mentioned above, mannose taken orally is rapidly excreted in the urine, and thus excreted mannose inhibits the binding of the bacteria to mannose lectin and cell surface sugar chains, thereby preventing urinary tract infections. Is expected.

  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.

  Chemical reaction methods and enzymatic methods are known as methods for preparing mannose.

  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. However, 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. However, 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. However, 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. However, 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. However, as with the fourth chemical reaction method, 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. However, there are problems in terms of the price of fructose as a raw material and the separation of fructose remaining in the reaction system.

  The second enzymatic method is a method of preparing mannose by reacting glucose or fructose with aldose-ketose isomerase (hereinafter referred to as "AKI"). However, this method is also problematic in terms of the separation of fructose remaining in the reaction system.

  In addition, for a sugar consisting of two or more sugar residues in which a reducing terminal sugar residue such as cellobiose is linked by a β-1,4 bond, the hydroxyl group at the 2-position of the reducing terminal sugar residue is 2-epimerylated Cellobiose 2-epimerase (Cellobiose 2-epimerase, hereinafter referred to as "CE") is known as an enzyme that catalyzes (isomerization) reaction or its reverse reaction (Patent Document 3, Patent Document 4). The enzyme can convert cellobiose in which glucose is β-1,4 linked to 4-O-β-glucosylmannose or reverse reaction thereof. In addition, Patent Document 4 describes that mannose is obtained when the cellobiose 2-epimerase is allowed to act on glucose (third enzyme method).

Japanese Patent Laid-Open No. 2001-213871 Japanese Patent Application Publication No. 2001-275583 JP 2012-130332 International Publication No. 2010/090095

However, in the third enzymatic method using CE described in Patent Document 4, in addition to the epimerization reaction, CE also catalyzes an aldose-ketose isomerization reaction. Therefore, fructose is also produced together with mannose, and there is a problem in separation of by-product fructose.
Also, until now, only AGE and CE have been reported as enzymes that isomerize between aldoses without substantially producing ketose, and enzymes performing similar reactions with unmodified monosaccharides are completely known so far. Not.

  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 which are presumed to encode N-acylglucosamine 2-epimerase (N-acylglucosamine 2-epimerase, hereinafter referred to as "AGE") on the genome Known (Gene Bank). As a result of expression analysis of each, two of the three had CE activity, but the remaining one did not have AGE activity and CE activity. With respect to the proteins which did not have this AGE activity and CE activity, their reactivities to various carbohydrates were examined. As a result, 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 (Mannose 2-epimerase, hereinafter referred to as "ME") which mutually converts glucose and mannose.

  Furthermore, a gene encoding a protein presumed to be AGE was cloned from Dyadobacter fermentans, and the reactivity of the recombinant enzyme to various carbohydrates was examined. As a result, it was revealed that among the proteins presumed to be AGE, there are proteins having ME activity.

  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 the 241 to 263 amino acid residues 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 with the amino acid sequence of the 264 to 423 amino acid residue of the amino acid sequence shown in and the glucose and mannose epimerization activity, 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: 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 amino acid sequence of the amino acid sequence of 263 to 419 shown in SEQ ID NO: 1, and having epimerization activity of glucose and mannose (However, SEQ ID NO: 2, SEQ ID NO: 4, except for the protein having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14).
[2]
The amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 263 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2,
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 at least 90% identity with the amino acid sequence of the 241 to 263 amino acid residues 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 the 241 to 263 amino acid residues of the amino acid sequence shown in SEQ ID NO: 4;
An amino acid sequence having an identity of 90% or more with the amino acid sequence of the 241 to 263 amino acid residues 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 of SEQ ID NO: 8 or amino acid sequence of amino acid residue 241 to 264 of the amino acid sequence of SEQ ID NO: 10 The protein according to [1], which is an amino acid sequence having an identity of 90% or more.
[3]
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: 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 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: 4;
An amino acid sequence having at least 90% identity to 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 with the amino acid sequence of the 264 to 423 amino acid residue 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 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 264-422 amino acid residue 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 The protein according to [1] or [2], which is an amino acid sequence having an identity of 90% or more.
[4]
An amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 262 amino acid residues 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,
An amino acid sequence having at least 90% identity with the amino acid sequence of amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence of 246 to 267 amino acid residue 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.
[5]
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 Amino acid sequence with 90% or more identity with
The amino acid sequence having 50% or more identity with the amino acid sequence of the amino acid sequence of the amino acid sequence of 263 to 419 of the amino acid sequence shown in SEQ ID NO: 12 is
An amino acid sequence having at least 90% identity with the amino acid sequence of the amino acid sequence of amino acid residue 263 to 419 of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence of amino acid residue 268 to 430 amino acid in the amino acid sequence shown in SEQ ID NO: 14 The protein according to [1] or [4], which is an amino acid sequence having an identity of 90% or more.
[6]
The epimerising agent of glucose and mannose containing the protein as described in any one of [1]-[5] as an active ingredient.
[7]
The protein according to any one of [1] to [5], further having epimerization activity of galactose and talose.
[8]
(C) has an insertion sequence of an amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 264 amino acid residues 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 on the N-terminal side of the inserted sequence, and SEQ ID NO: 10 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 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 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 amino acid sequence of amino acid sequence 263 to 419 of the amino acid sequence shown in and an epimerylation activity of galactose and talose (However, SEQ ID NO: 2, SEQ ID NO: 4, except for the protein having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14).
[9]
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 the 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 to 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 the 265 to 428 amino acid residue of the amino acid sequence shown in SEQ ID NO: 10 is the amino acid sequence of the 265 to 428 amino acid residue of the amino acid sequence shown in SEQ ID NO: 10 The protein according to [8], which is an amino acid sequence having an identity of 90% or more.
[10]
An amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 262 amino acid residues 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,
An amino acid sequence having at least 90% identity with the amino acid sequence of amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence of 246 to 267 amino acid residue 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
An amino acid sequence having 90% 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 or an amino acid sequence 1 to 245 amino acid residue of the amino acid sequence shown in SEQ ID NO: 14 90% or more of the amino acid sequence
An amino acid sequence having an identity of 50% or more with the amino acid sequence of the 263-419 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 is the amino acid sequence of the 263-419 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 The amino acid sequence having an identity of 90% or more, or an amino acid sequence having an identity of 90% or more with the amino acid sequence of the 268 to 430 amino acid residues of the amino acid sequence shown in SEQ ID NO: 14; protein.
[11]
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 an amino acid sequence of the protein having 70% or more identity 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.
[12]
From proteins not known to have glucose and mannose epimerization activity and galactose and talose epimerization activity (a) identical to the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2 Select a protein having an amino acid sequence of 70% or more, or (b) a protein having an amino acid sequence of 70% or more identity to the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 And (c) selecting a protein having an epimerylation activity of glucose and mannose, an epimerylation activity of galactose and talose, or both from the proteins selected in (c) (a) or (b),
A method for producing a protein having an epimerization activity of glucose and mannose and / or an epimerization activity of galactose and talose.
[13]
(D) Amino acid sequence The amino acid sequence of a known or unknown protein has 70% or more identity with the amino acid sequence of amino acid sequence 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 with the amino acid sequence of the 241 to 263 amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of the 241 to 262 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 To an amino acid sequence having an identity of 70% or more, and (f) (d) or (e) Includes a modified protein, epimerization activity of glucose and mannose, the selection of proteins with epimerization activity or both galactose and talose,
Method for producing a protein having epimerization activity
[14]
The amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 263 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2,
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 at least 90% identity with the amino acid sequence of the 241 to 263 amino acid residues 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 the 241 to 263 amino acid residues of the amino acid sequence shown in SEQ ID NO: 4;
An amino acid sequence having an identity of 90% or more with the amino acid sequence of the 241 to 263 amino acid residues 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 of SEQ ID NO: 8 or amino acid sequence of amino acid residue 241 to 264 of the amino acid sequence of SEQ ID NO: 10 The method according to [12] or [13], wherein the amino acid sequence is 90% or more.
[15]
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 of (a), (d) or (e);
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: 4;
An amino acid sequence having at least 90% identity to 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 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 264-422 amino acid residue 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 The method according to any one of [12] to [14], further having an amino acid sequence having an identity of 90% or more.
[16]
An amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 262 amino acid residues 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,
An amino acid sequence having at least 90% identity with the amino acid sequence of amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence of 246 to 267 amino acid residue 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.
[17]
(B), (d) or (e), an amino acid sequence having 50% or more or 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 SEQ ID NO: 14 further has an amino acid sequence having 90% or more identity with the amino acid sequence of 1 to 245 amino acid residues of the amino acid sequence shown in SEQ ID NO: 14 and Amino acid sequence having at least 50% or at least 90% identity with the amino acid sequence of the group, or at least 90% identity with the amino acid sequence of 268 to 430 amino acid residues of the amino acid sequence shown in SEQ ID NO: 14 The production method according to [12], [13] or [16], further comprising.
[18]
The production method according to any one of [12] to [17], which is a production of a protein having an epimerization activity of glucose and mannose.
[19]
The production method according to any one of [12] to [17], which is production of a protein having epimerization activity of galactose and talose.
[20]
The method according to any one of [1] to [5], the protein according to any one of [1] to [5], the epimellitic agent according to [6] or [11], or the method according to any one of [12] to [18] A method for producing mannose, which comprises causing the protein produced in claim 1 to act to produce mannose.
[21]
A galactose containing as an active ingredient a protein having the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14 or a protein having galactose and talose epimerization activity of 70% or more identity to the amino acid sequence of the protein And Tulose epimerising agent.
[22]
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 epimerization according to [21] in galactose A method for producing talose, which comprises causing an agent to act to produce talose.

  According to 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 reaction test result with respect to various substrates of Runsl 4512 gene product (RsME) is shown. [Figure 6] Figure 6 shows the results of the reactivity test of RsME against glucose, mannose and fructose. The test result which calculates | requires the optimal pH of RsME is shown. The pH stability test result of RsME is shown. The heat stability test result of RsME is shown. The mannose production test (temperature dependence) result from glucose of RsME is shown. The reaction test result with respect to various substrates of Dfer — 5652 gene product (DfME) is shown. The reaction test result with respect to various substrates of DfME is shown. The reaction test result with respect to various substrates of DfME is shown. The test result which calculates | requires the optimal pH of DfME is shown. 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. Comparison table with a primary amino acid sequence near the insertion sequence, which is a protein of unknown function, including the protein Dfer — 5652. 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 description of carbohydrate in the present specification refers to D-form unless otherwise specified.

<Proteins having epimerization activity of glucose and mannose>
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 the 241 to 263 amino acid residues 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 residues of the amino acid sequence shown in and a protein having an epimerization activity of glucose and mannose.
(B) an insertion sequence of an amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 262 amino acid residues 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 on the N terminal side of the inserted sequence, and SEQ ID NO: 12 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 amino acid sequence of the amino acid sequence of 263 to 419 shown in the above, and having an epimerization activity of glucose and mannose.

  As mentioned above, it is known that Runella slithyformis (DSM19594) has three genes presumed to encode N-acylglucosamine 2-epimerase (AGE) on the genome ( Gene Bank). As a result of expression analysis of each of the present inventors, two of the three had CE activity, but the remaining one did not have AGE activity and CE activity. With respect to the protein which did not have this AGE activity and CE activity, the reactivity to various carbohydrates was examined (Example 1). As a result, 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. 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). However, 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.

  In 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 base sequence of the Dfer_ 5652 gene is the base sequence of SEQ ID NO: 3, and from this base sequence, the protein (Dfer_ 5652) consists of the amino acid sequence of 1 to 423 amino acid residues represented by 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.

  These two proteins 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. 23 a-c), and this insertion 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 241 to 263 amino acid residues 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 identity with a certain degree or more with this insertion sequence, and 5 additional protein functions It was investigated. They are, for example, Emtisticia oligotrophica (Emticicia oligotrophica), Spirosoma ringgare (Spirosoma linguale), Sedimini spilocator Semaradinae (Sediminispirochaeta smaragdinae), Treponema brennaborens (Treponema brennaborense) and 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. As a result, it was revealed that all proteins presumed to be AGE or the like having an insertion sequence are proteins having ME activity (Emtol_1243, Slin_6381, Spirs_3060, Trebr_2076 and Treaz_2550) (Examples 3 to 7). 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) 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.

The 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. Furthermore, an amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 263 amino acid residues 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 to 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) 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 the 241 to 263 amino acid residues of the amino acid sequence shown in SEQ ID NO: 4;
An amino acid sequence having an identity of 90% or more with the amino acid sequence of the 241 to 263 amino acid residues 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 of SEQ ID NO: 8 or amino acid sequence of amino acid residue 241 to 264 of the amino acid sequence of SEQ ID NO: 10 And an amino acid sequence having an identity of 90% or more. These 241 to 263 amino acid residues (SEQ ID NO: 2, 4, 6, 8) or 241 to 264 amino acid residues (SEQ ID NO: 10) are insertion sequences in each protein.

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: 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. Furthermore, 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 (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 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: 4;
An amino acid sequence having at least 90% identity to 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.

The 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. Furthermore, 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 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 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 264-422 amino acid residue 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.

Preferably, the amino acid sequence having 70% or more identity with the amino acid sequence of the 241 to 262 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (b) 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 an identity of 70% or more with the amino acid sequence of the 241 to 262 amino acid residue 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 an identity of 70% or more with the amino acid sequence of the 241 to 262 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (b) is the 241 to 262 amino acid sequence of the amino acid sequence An amino acid sequence having at least 90% identity with the amino acid sequence of the amino acid residue, or at least 90% identity with the amino acid sequence of the 246-267 amino acid residue of the amino acid sequence shown in SEQ ID NO: 14 You can also.

  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: 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. Furthermore, 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 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 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.

  The amino acid sequence having an identity of 50% or more with the amino acid sequence of the amino acid sequence of the amino acid residue of 263 to 419 in the protein of SEQ ID NO: 12 in the protein of (b) preferably has an identity of 60% or more, 70% or more 80% or more, 90% or more, or 95% or more. Furthermore, an amino acid sequence having 50% or more identity to the amino acid sequence of the amino acid sequence of amino acid residues 263 to 419 of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (b) Amino acid sequence having at least 90% identity with the amino acid sequence of the 263-419 amino acid residue, or at least 90% identity with the amino acid sequence of 268-430 amino acid residue of the amino acid sequence shown in SEQ ID NO: 14 Can be.

  In the present specification, 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.

In the present invention, 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
The enzyme reaction is stopped by holding 100 μL 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 . The amount of glucose contained in 50 μL 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 μmol 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
The enzyme reaction is stopped by holding 100 μL 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 . 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 μmol talose per minute.

<Epimelizing agent>
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.

<Proteins having epimerization activity of galactose and talose>
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 the 241 to 264 amino acid residues 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 on the N-terminal side of the inserted sequence, and SEQ ID NO: 10 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 265 to 428 amino acid residue of the amino acid sequence shown in and a protein having an epimerylation activity of galactose and talose.
(D) having an insertion sequence of an amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 262 amino acid residues 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 amino acid sequence of 263 to 419 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 residue 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 an identity of 90% or more 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 to 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, for example, 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 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, an 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) is the amino acid sequence of 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.

The amino acid sequence having 70% or more identity with the amino acid sequence of the 241 to 262 amino acid residue 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. Furthermore, an 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) is a compound of the 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,
An amino acid sequence having at least 90% identity with the amino acid sequence of amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence of 246 to 267 amino acid residue 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 (1-430 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.

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: 12 in the protein (d) 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 to 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,
An amino acid sequence having 90% 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 or an 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 amino acid sequence of the amino acid residue of 263 to 419 in the protein of (d) shown in SEQ ID NO: 12 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 to the amino acid sequence of the amino acid sequence of the amino acid residue of 263 to 419 of the amino acid sequence shown in SEQ ID NO: 12 in the protein of (d) Amino acid sequence having an identity of 90% or more with the amino acid sequence of the amino acid residue of 263-419, or an amino acid having an identity of 90% or more with the amino acid sequence of amino acid residue 268-430 of the amino acid sequence shown in It can be an array.

  The origin of the protein of the present invention is not particularly limited. For example, 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.

  When producing the recombinant protein of the present invention by genetic engineering technology, 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. In 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. . As usable expression vectors, it is possible to use various 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. Alternatively, 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.

  When the protein of the present invention is expressed in an expression system using a specific species as a host, 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.

  In the present invention, a 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. For example, as described in the following examples, 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. Also, 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. In addition to such production methods, the present invention also includes the following production methods (1) and (2).

<Method for producing protein having epimerization activity>
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 of 70% or more identity to the amino acid sequence of the 241 to 262 amino acid residues 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. As a protein of which the amino acid sequence is known for use in the present production method, a protein having an insertion sequence shown in the following Table 5 and FIGS. 23b and 23c can be exemplified. From the selected protein in (c), 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 according to the present invention comprises (d) the amino acid sequence of the known or unknown amino acid sequence of SEQ ID NO: 2 An amino acid sequence having 70% or more identity with the amino acid sequence of the 241-263 amino acid residue of the amino acid sequence shown, or an amino acid sequence with the amino acid sequence of the 241-262 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 % Or more of the amino acid sequence, or (e) amino acid sequence amino acid sequence of at least part 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 Modifying the 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, and from the protein inserted or modified in (f) (d) or (e), glucose and mannose And selecting a protein having an epimerization activity of galactose, an epimerization activity of galactose and talose, or both.

  The 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. As an example of a protein whose amino acid sequence is known, the protein shown in FIG. 23a can be mentioned. From the inserted and modified proteins, 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. The 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. Preferably, 80% or more, 90% or more, 95% or more, 98%, from the viewpoint that the probability of selecting a protein having an epimerization activity of glucose and mannose, an epimerization activity of galactose and talose, or both is increased. It is more preferable to perform sequence insertion and / or modification on the amino acid sequence of the protein which is the above or 99% or more. Moreover, the 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. 23a, and 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. By 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.

In the methods (1) and (2) for producing a protein having an epimerization activity of glucose and mannose and / or an epimerization activity of galactose and talose,
Preferably, the 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 (a), (d) or (e) preferably has an identity of 80 % Or more, 90% or more, 95% or more, 98% or more, or 99% or more, for example, 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), the identity of the amino acid sequence of amino acid sequence 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2 90% or more amino acid sequence,
An amino acid sequence having at least 90% identity to the amino acid sequence of the 241 to 263 amino acid residues of the amino acid sequence shown in SEQ ID NO: 4;
An amino acid sequence having an identity of 90% or more with the amino acid sequence of the 241 to 263 amino acid residues 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 of SEQ ID NO: 8 or amino acid sequence of amino acid residue 241 to 264 of the amino acid sequence of SEQ ID NO: 10 And an amino acid sequence having an identity of 90% or more.

In the methods (1) and (2) for producing a protein having epimerization activity,
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 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: 4;
An amino acid sequence having at least 90% identity to 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 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 264-422 amino acid residue 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 a protein further having an amino acid sequence of 90% or more.

In the methods (1) and (2) for producing a protein having epimerization activity,
Preferably, the amino acid sequence having 70% or more identity with the amino acid sequence of the 241 to 262 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 in (b), (d) or (e) preferably has an identity of 80 %, 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,
An amino acid sequence having at least 90% identity with the amino acid sequence of amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence of 246 to 267 amino acid residue of the amino acid sequence shown in SEQ ID NO: 14 And an amino acid sequence having an identity of 90% or more.

In the methods (1) and (2) for producing a protein having epimerization activity,
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 an identity of 50% or more or 90% or more with the amino acid sequence of the group, or an amino acid sequence with 90% or more identity 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 amino acid sequence of the amino acid sequence of SEQ ID NO: 12 or the amino acid sequence of SEQ ID NO: 14 The protein may further have an amino acid sequence of 90% or more identity to the amino acid sequence of the 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. In the 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.

<Manufacturing method of mannose>
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 (mannose containing carbohydrate composition) can be manufactured by adding the protein of this invention to the said solution, and maintaining at 30-60 degreeC 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.

  Although a pure glucose product may be used as a substrate for producing mannose from glucose, a sugar composition containing other carbohydrates may be used from the viewpoint of cost etc. For example, starch syrup, powdered starch, It is possible to use, for example, hydrol (liquid separation produced during crystalline glucose production). Furthermore, for example, 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.

<Epemimelizing agent of galactose and talose>
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 Preferably, 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.

<Production method of talose>
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. The production of talose from galactose using the protein etc. of the present invention is carried out, for example, by preparing an aqueous solution of carbohydrate having a solid content concentration of about 1 to 50% containing galactose, and using pH of 6 to 10 using acid or alkali as necessary. By adjusting to a certain extent, the protein of the present invention is added to the solution and maintained at 30 to 60 ° C. for 1 to 72 hours, thereby producing talose (a talose-containing carbohydrate composition). After the above enzyme reaction, if necessary, the talose-containing carbohydrate composition can be subjected to a decolorization, desalting, purification treatment and the like using a conventional method. Furthermore, by subjecting the obtained talose-containing carbohydrate composition to a resin fractionation treatment or the like, talose and the galactose which is a substrate can be separated to increase the talose purity. In addition, talose (a talose-containing carbohydrate composition) 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. Furthermore, for example, 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.

  Hereinafter, the present invention will be described in more detail based on examples. However, the examples are illustrative of the present invention, and the present invention is not intended to be limited to the examples.

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.

(1) Cloning of Runsl 4512 gene The 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.

(2) Preparation of Runsl — 4512 Gene Product The Runsl — 4512 gene shown in SEQ ID NO: 1 from which the termination codon had been 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 μg / 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. After ice-cooling the culture solution, 1 mL of 0.1 mol / L isopropyl β-thiogalactopyranoside was added to a final concentration of 0.1 mmol / L, and the culture was performed at 16 ° C. for 24 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 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. Samples were analyzed by SDS-PAGE to confirm a single band.

(3) TLC analysis of the reaction product 5 μL of the reaction solution consisting of 50 mM various substrates, 50 mM sodium phosphate buffer (pH 7) and 0.172 mg / mL Runsl 4512 is kept at 37 ° C. for 4 hours, and 1 μL of this The mixture was subjected to TLC using butanol / 2-propanol / water = 2/2/1 as a developing solvent. The sugar was detected by heating anisaldehyde / sulfuric acid / acetic acid = 1/2/97 on a TLC plate and heating. D-glucose, D-mannose, D-glucosamine, N-acetylglucosamine, D-allose, D-galactose, D-xylose, L-fucose, L-arabinose, L-rhamnose, D-glucose 6-phosphate, D -Mannose 6-phosphate, D-fructose, D-lyxose, D-tagatose, D-xylulose, L-sorbose, D-talose, D-altulose, β-1,4-mannobiose and D-gluconic acid as substrates And The results are shown in FIG. Reaction products were detected only from D-glucose and D-mannose.

  For reactions on D-glucose, D-mannose and D-fructose, samples before and after the reaction were analyzed by TLC. The reaction solution (20 μL) having the same composition as the above was similarly reacted, and subjected to TLC. The results are shown in FIG. The formation of D-mannose from D-glucose and the formation of D-glucose from D-mannose were confirmed. On the other hand, no reaction product was detected from D-fructose. Thus, Runsl 4512 was judged to be mannose 2-epimerase, which catalyzes the interconversion of D-glucose and D-mannose.

Activity measurement
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 μL 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 μmol 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.

Kinetic parameters for mannose
The enzyme reaction was terminated by holding the reaction solution containing 10-100 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 reaction rate obtained was regressed to the rate equation of Michaelis Mentene to obtain K _m 55.8 mM, k _cat 4.55 s ^-1 .

Temporal change of reaction to mannose
Hold 1 mL of a reaction solution consisting of 0.1 M mannose, 40 mM Tris-HCl buffer (pH 7.8), and enzyme at 37 ° C, collect 50 μL of the reaction solution as appropriate, and hold at 80 ° C for 3 minutes to stop the reaction did. The glucose concentration was quantified by the method described above. The glucose concentration at 72 hours of reaction was 72 mM.

Production of mannose from glucose
4.2-16.6 μM Runsl_4512, holding 0.5 mL of a reaction solution consisting of 40 mM Tris-HCl (pH 7.8) and 30% glucose at 35-55 ° C, collecting a 0.1 mL sample after 24 hours, and keeping at 85 ° C for 10 minutes The reaction was stopped by heating. This was diluted 6-fold and 10 μL was subjected to HPLC. In HPLC, Sugar D (Nakalai) was used for the column, the column temperature was 30 ° C., the mobile phase was 80% acetonitrile, and the flow rate was 1 mL / min. Sugar was detected by RI detector. The results are shown in FIG. Under the reaction conditions of enzyme concentration 8.3 μM or more and 50 ° C. or less, the mannose concentration after the reaction was almost constant and became 7%. The conversion rate of glucose to mannose was 23%.

Example 2
In this example, 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.

(1) 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.

(2) Preparation of Dfer — 5652 Gene Product The 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 μg / 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. After ice-cooling the culture solution, 1 mL of 0.1 mol / L isopropyl β-thiogalactopyranoside was added to a final concentration of 0.1 mmol / L, and the culture was performed 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. Samples were analyzed by SDS-PAGE to confirm a single band.

TLC analysis of reaction products
Five μL 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 μL of this was subjected to TLC. The developing solvent is shown below. The sugar was detected by injecting anisaldehyde / sulfuric acid / acetic acid = 1/2/97 onto the TLC plate after development and heating. D-glucose, D-mannose, L-rhamnose, D-talose, D-altose, D-galactose, L-fucose, L-fructose, D-tagatose, L-sorbose, D-lyxose, L-arabinose as a substrate , D-xylose, D-xylulose, N-acetyl-D-glucosamine, β-1,4-mannobiose, D-glucosamine and D-gluconic acid, reaction products only from D-glucose and D-mannose Was detected.

The developing solvent used was different for each substrate.
2-propanol / 1-butanol / water = 2/2/1
D-glucose, D-mannose, D-rhamnose, D-talose, D-altrose, D-galactose, L-fucose, D-fructose, D-tagatose, L-sorbose, D-lyquise, L-arabinose, D -Xylose, D-xylulose, N-acetyl-D-glucosamine, β-1,4-mannobiose
1-butanol / acetic acid / water = 8/3/2 D-glucosamine
2-propanol / 1-butanol / water = 12/3/4 D-gluconic acid

  The results are shown in FIG. 7, FIG. 8 and FIG. The results of the substrate test confirmed that D-glucose produces D-mannose and D-mannose produces D-glucose. Dfer_ 5652 catalyzes the interconversion of D-glucose and D-mannose. It was determined that

Activity measurement
The enzyme reaction was terminated by holding 100 μL of a reaction solution containing 10 mM mannose, 40 mM HEPES-NaOH buffer (pH 8.0), and the enzyme at 37 ° C. for 20 minutes and heating at 80 ° C. for 5 minutes. The amount of glucose contained in 50 μL of the supernatant obtained by centrifugation at 12000 rpm for 10 minutes was quantified by the hexokinase method using Thio-NAD ⁺ . That 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 dehydrogenase ( An equal amount of a color developing reagent containing Nacalai Tesque is added to the reaction solution and kept at 37 ° C. for 20 minutes, and the absorbance of Thio-NADH produced along with the G6P oxidation reaction is measured by a spectrophotometer Glucose in the reaction mixture was quantified. The specific activity of Dfer — 5652 obtained under the reaction conditions was 0.688 U / mg. Here, the enzyme activity of 1 U was defined as the amount of enzyme that produces 1 μmol of glucose per minute.

Optimal pH
The enzyme reaction was terminated by holding 100 μL 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 μL 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 microliters of enzyme solutions were hold | maintained for 20 minutes at 20-55 degreeC, and activity was measured in the procedure similar to the said activity measurement. The results are shown in FIG. The activity of 80% or more was maintained at 45 ° C. or less.

Kinetic parameters for mannose
Maintain 100 μL of the reaction solution containing 4-150 mM mannose, 40 mM HEPES-NaOH buffer (pH 8.0) and enzyme at 37 ° C, and heat the reaction at 80 ° C for 5 minutes for 7 minutes and 14 minutes. Stopped the enzyme reaction. When the obtained reaction rate was plotted, a saturation curve was obtained. Regression to Michaelis Mentene's velocity equation gave K _m 66.4 mM, k _cat 3.88 s ^-1 .

  When the amino acid sequences of AGE, CE and AKI of which function is known for Runsl 4512 and Dfer_ 5652 confirmed to be ME in Example 1 and Example 2 are compared, the above two MEs are amino acid sequences of proteins of which function is known It turned out that it has an insertion sequence in specific site compared with (Fig. 23 ac). The insertion sequence is considered to be important for the function of ME, and the function was confirmed for five types of proteins of unknown function that have the insertion sequence similarly estimated on the AGE etc. database.

[Examples 3 to 7]
In this example, Emtitissia oligotrophica (Emticicia oligotrophica), Spirosoma ringgare (Spirosoma linguale), Sedimini spilocator Smarajinae (Sedimini pirochaeta smaragdinae), Treponema brenabolens (Treponema brennaborense) and tropemeite. Genes estimated on the database with AGE etc. (each encoding Emtol_1243, Slin_6381, Spirs_3060, Trebr_2067 and Treaz_2550, respectively) are optimized by combining with the codon usage frequency of E. coli. And produced as a recombinant enzyme and characterized for gene products.

Synthesis of Target Gene The 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.

Preparation of expression plasmid
In order to investigate expression in E. coli, the target gene purchased was subcloned between the NdeI site of pET23a and the histidine tag. PCR was performed using pET23a or pEX-A2J1 into which the target gene was inserted as a template to amplify the linearized pET23a and the target gene excluding the termination codon. PrimeSTAR Max DNA Polymerase (Takara Bio) was used for PCR. The obtained PCR reaction solution was subjected to agarose gel electrophoresis, a band present around the desired size was cut out from the gel after electrophoresis, and the PCR amplification product was extracted from the gel after cut out. The target gene was inserted into pET23a using In-Fusion HD Cloning Kit (Takara Bio). E. coli DH5α 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.

Expression in E. coli
The expression plasmid was introduced into 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. When the culture was performed at 37 ° C. and 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.

Extraction of Recombinant Enzymes 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 μm 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. That is, after washing the carrier with the above buffer, it was eluted with 500 mM imidazole-HCl buffer (pH 7) containing 0.5 M NaCl. The eluted fraction was collected and concentrated using Amicon Ultra until the solution volume reached 15 mL. The obtained sample was dialyzed into 20 mM HEPES-NaOH buffer (pH 7). It filtered with a 0.45 micrometer filter, and used the filtrate as a sample for subsequent analysis. The protein concentration of the sample was determined by multiplying the measured specific absorbance from the amino acid sequence by A280 measured.

SDS-PAGE
The same amount of sample buffer was added to 7.5 μL of each sample diluted 10-fold with DW, and the whole was heated at 99.9 ° C. for 5 minutes and subjected to SDS-PAGE. confirmed.

Activity measurement
The enzyme reaction was stopped by holding 100 μL of a reaction solution containing 10 mM mannose, 40 mM HEPES-NaOH buffer (pH 8.0), and enzyme at 37 ° C for 30 minutes or 60 minutes, and heating at 80 ° C for 5 minutes . The amount of glucose contained in 50 μL of the supernatant obtained by centrifugation at 12000 rpm for 10 minutes was quantified by the hexokinase method using Thio-NAD ⁺ . That is, 1 mM Thio-NAD ⁺ , 7 mM ATP, 0.1 M Tris-HCl (pH 7.5), 1.0 mM MgCl ₂ , 2.0 U / mL hexokinase (from baker's yeast, nacalai tesque) and 2.0 U / mL G6P dehydrogenase ( An equal amount of a coloring reagent containing 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 measured by a spectrophotometer Glucose in the reaction mixture was quantified. The amount of enzyme producing 1 μmol glucose per minute was defined as 1 U. The results are shown in Table 1.

Examination of substrate specificity of Emtol_1243, Spirs_3060, Slin_6381, Trebr_2067, Treaz_2550 (TLC)
A 5 μL reaction solution containing 50 mm various substrates, 5 mm HEPES-NaOH (pH 8.0) 0.5 mg / mL BSA and an enzyme was kept at 37 ° C. for 24 hours, and 1 μL was spotted on a TLC plate. The TLC plate after developing with each developing solvent was sprayed with anisaldehyde / sulfuric acid / acetic acid = 1/2/97 and heated to detect sugar. The enzyme concentration in the reaction solution is shown below. 8.0 μm Emtol_1243, 9.5 μm Spirs_3060, 9.5 μm Slin_6381, 10 μm Trebr_2067, 9.5 μm Treaz_2550. As a substrate, D-glucose, D-mannose, D-fructose, D-galactose, D-talose, D-allose, D-altrose, L-sorbose, D-tagatose, L-arabinose, D-xylose, D- Lyxose, L-rhamnose, L-fucose, cellobiose, β- (1,4) glucosyl mannose, β- (1,4) mannosyl glucose, β- (1,4) mannobiose, lactose, epilactose, lactulose, sucrose, Maltose, N-acetyl D-glucosamine was used. As developing solvents, 2-propanol / 1-butanol / water = 2/2/1 solution for monosaccharide and N-acetyl d-glucosamine, 2-propanol / 1-butanol / water = 12/3 for disaccharide / 4 solutions were used respectively.
The results are shown below in FIGS. In any of the figures, B: Blank (inactivation treated enzyme added zone), R: Reaction (enzyme added zone) is meant.

  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.

Those in which the reactive organisms were identified were indicated by +, those not identified by-, and those not tested were indicated in blanks.

Compare the amino acid sequences of the above 7 enzymes confirmed to have ME activity with the amino acid sequences of known AGEs and CE, etc. and the amino acid sequences of proteins presumed to be AGE etc. of other unknown functions, and compare the inserted sequence part The results are shown in Figures 23a-c. In addition, the supplementary information regarding the function known enzyme shown by FIG. 23 a is as follows.
* 1: AGE derived from Porcine kidney (FEBS Letters 587 (2013) 840-846)
* 2: AGE derived from Anabaena sp. (FEBS Letters 587 (2013) 840-846)
* 3: CE from Rhodothermus marinus (JP 2012-130332)
* 4: CE from Ruminococcus albus (Appl. Microbiol. Biotechnol. (2008) 79, 433-441)
* 5: CE from Caldicellulosiruptor saccharolyticus (WO 2010/090095)
* 6: AKI from Escherichia coli (J. Mol. Biol. (2008) 377, 1443-1459)
* 7: AKI from Salmonella enteritica (J. Mol. Biol. (2008) 377, 1443-1459)
The insertion sequences of the amino acid sequences of the above seven enzymes are shown in Table 3. In addition, the identity of the amino acid sequence of Runsl 4512 shown in SEQ ID NO: 2 with the amino acid sequence of the other 6 enzymes and the identity of the amino acid sequence of Trebr 2067 shown in SEQ ID NO: 12 with the amino acid sequence of the other 6 enzymes are shown in Table 4. The

  It was revealed that even those enzymes having an insertion sequence less than 40% identical to the amino acid sequence of Runsl 4512 have the same ME activity. Also for the proteins with known and unknown functions shown in FIG. 23 a and Table 5, those having the same insertion sequence may have ME activity, and further, shown in FIGS. 23 b and 23 c and Table 7 Table 7 With respect to proteins of unknown function, those having the same insertion sequence are strongly presumed to have ME activity, and the method for producing a protein having epimerization activity of glucose and mannose and / or epimerization activity of galactose and talose according to the present invention The protein having epimerization activity can be produced as a protein not known to have the epimerization activity of glucose and mannose and the epimerization activity of galactose and talose.

Significance of Insertion Sequence From the homology of primary amino acid sequences, the enzyme of the present invention is presumed to be similar in three-dimensional structure to cellobiose 2-epimerase having an α / α-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 α7 helix and the α8 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_3481 base sequence No. 8: Slin_6381 amino acid sequence 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. 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

Claims (22)

(A) has an insertion sequence of an amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 263 amino acid residues 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 with the amino acid sequence of the 264 to 423 amino acid residue of the amino acid sequence shown in and the glucose and mannose epimerization activity, 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: 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 amino acid sequence of the amino acid sequence of 263 to 419 shown in SEQ ID NO: 1, and having epimerization activity of glucose and mannose (However, SEQ ID NO: 2, SEQ ID NO: 4, except for the protein having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14). The amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 263 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2,
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 at least 90% identity with the amino acid sequence of the 241 to 263 amino acid residues 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 the 241 to 263 amino acid residues of the amino acid sequence shown in SEQ ID NO: 4;
An amino acid sequence having an identity of 90% or more with the amino acid sequence of the 241 to 263 amino acid residues 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 of SEQ ID NO: 8 or amino acid sequence of amino acid residue 241 to 264 of the amino acid sequence of SEQ ID NO: 10 The protein according to claim 1, which is an amino acid sequence having an identity of 90% 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: 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 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: 4;
An amino acid sequence having at least 90% identity to 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 with the amino acid sequence of the 264 to 423 amino acid residue 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 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 264-422 amino acid residue 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 The protein according to claim 1 or 2, wherein the amino acid sequence is 90% or more. An amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 262 amino acid residues 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,
An amino acid sequence having at least 90% identity with the amino acid sequence of amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence of 246 to 267 amino acid residue of the amino acid sequence shown in SEQ ID NO: 14 The protein according to claim 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 Amino acid sequence with 90% or more identity with
The amino acid sequence having 50% or more identity with the amino acid sequence of the amino acid sequence of the amino acid sequence of 263 to 419 of the amino acid sequence shown in SEQ ID NO: 12 is
An amino acid sequence having at least 90% identity with the amino acid sequence of the amino acid sequence of amino acid residue 263 to 419 of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence of amino acid residue 268 to 430 amino acid in the amino acid sequence shown in SEQ ID NO: 14 The protein according to claim 1 or 4, wherein the amino acid sequence is 90% or more. An epimerising agent for glucose and mannose comprising the protein according to any one of claims 1 to 5 as an active ingredient. The protein according to any one of claims 1 to 5, further having an epimerization activity of galactose and talose. (C) has an insertion sequence of an amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 264 amino acid residues 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 on the N terminal side of the inserted sequence, and SEQ ID NO: 10 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 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 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 amino acid sequence of amino acid sequence 263 to 419 of the amino acid sequence shown in and an epimerylation activity of galactose and talose (However, SEQ ID NO: 2, SEQ ID NO: 4, except for the protein having the amino acid sequence shown in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14). 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 the 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 to 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 the 265 to 428 amino acid residue of the amino acid sequence shown in SEQ ID NO: 10 is the amino acid sequence of the 265 to 428 amino acid residue of the amino acid sequence shown in SEQ ID NO: 10 The protein according to claim 8, which is an amino acid sequence having an identity of 90% or more. An amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 262 amino acid residues 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,
An amino acid sequence having at least 90% identity with the amino acid sequence of amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence of 246 to 267 amino acid residue 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
An amino acid sequence having 90% 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 or an amino acid sequence 1 to 245 amino acid residue of the amino acid sequence shown in SEQ ID NO: 14 90% or more of the amino acid sequence
An amino acid sequence having an identity of 50% or more with the amino acid sequence of the 263-419 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 is the amino acid sequence of the 263-419 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 The amino acid sequence having an identity of 90% or more, or an amino acid sequence having an identity of 90% or more with an amino acid sequence of 268 to 430 amino acid residues of the amino acid sequence shown in SEQ ID NO: 14. protein. 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 an amino acid sequence of the protein having 70% or more identity 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. From proteins not known to have glucose and mannose epimerization activity and galactose and talose epimerization activity (a) identical to the amino acid sequence of amino acid residues 241 to 263 of the amino acid sequence shown in SEQ ID NO: 2 Select a protein having an amino acid sequence of 70% or more, or (b) a protein having an amino acid sequence of 70% or more identity to the amino acid sequence of amino acid residues 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 And (c) selecting a protein having an epimerylation activity of glucose and mannose, an epimerylation activity of galactose and talose, or both from the proteins selected in (c) (a) or (b),
A method for producing a protein having an epimerization activity of glucose and mannose and / or an epimerization activity of galactose and talose. (D) Amino acid sequence The amino acid sequence of a known or unknown protein has 70% or more identity with the amino acid sequence of amino acid sequence 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 with the amino acid sequence of the 241 to 263 amino acid residue of the amino acid sequence shown in SEQ ID NO: 2 or the amino acid sequence of the 241 to 262 amino acid residue of the amino acid sequence shown in SEQ ID NO: 12 To an amino acid sequence having an identity of 70% or more, and (f) (d) or (e) Includes a modified protein, epimerization activity of glucose and mannose, the selection of proteins with epimerization activity or both galactose and talose,
Method for producing a protein having epimerization activity The amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 263 amino acid residues of the amino acid sequence shown in SEQ ID NO: 2,
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 at least 90% identity with the amino acid sequence of the 241 to 263 amino acid residues 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 the 241 to 263 amino acid residues of the amino acid sequence shown in SEQ ID NO: 4;
An amino acid sequence having an identity of 90% or more with the amino acid sequence of the 241 to 263 amino acid residues 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 of SEQ ID NO: 8 or amino acid sequence of amino acid residue 241 to 264 of the amino acid sequence of SEQ ID NO: 10 The production method according to claim 12 or 13, wherein the amino acid sequence is 90% or more. 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 of (a), (d) or (e);
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: 4;
An amino acid sequence having at least 90% identity to 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 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 264-422 amino acid residue 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 The production method according to any one of claims 12 to 14, further comprising an amino acid sequence having an identity of 90% or more. An amino acid sequence having an identity of 70% or more with the amino acid sequence of the 241 to 262 amino acid residues 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,
An amino acid sequence having at least 90% identity with the amino acid sequence of amino acid sequence 241 to 262 of the amino acid sequence shown in SEQ ID NO: 12 or the amino acid sequence of 246 to 267 amino acid residue of the amino acid sequence shown in SEQ ID NO: 14 The production method according to claim 12 or 13, wherein the amino acid sequence is 90% or more. (B), (d) or (e), an amino acid sequence having 50% or more or 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 SEQ ID NO: 14 further has an amino acid sequence having 90% or more identity with the amino acid sequence of 1 to 245 amino acid residues of the amino acid sequence shown in SEQ ID NO: 14, and residues 263 to 419 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% identity with the amino acid sequence of 268 to 430 amino acid residues of the amino acid sequence shown in SEQ ID NO: 14 The method according to claim 12, 13 or 16, further comprising. The production method according to any one of claims 12 to 17, which is a production of a protein having an epimerization activity of glucose and mannose. The production method according to any one of claims 12 to 17, which is production of a protein having epimerization activity of galactose and talose. The protein according to any one of claims 1 to 5, the epimellitic agent according to any one of claims 6 or 11, or the protein produced by the method according to any one of claims 12 to 18 acting on glucose A process for producing mannose, comprising: producing mannose. A galactose containing as an active ingredient a protein having the amino acid sequence shown in SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14 or a protein having galactose and talose epimerization activity of 70% or more identity to the amino acid sequence of the protein And Tulose epimerising agent. Allowing galactose to act on the protein according to any one of claims 7 to 10, the protein produced by the method according to any one of claims 12 to 17, or the epimerising agent according to claim 21 A method for producing talose, comprising producing talose.