JP7592539B2 - Enzyme preparation for catalyzing epimerization reaction of sugar, method for producing epimerization reaction product, and epimerization reaction product - Google Patents

Description

The present invention relates to an enzyme agent for catalyzing the epimerization reaction of sugars, a method for producing an epimerization reaction product, which includes a step of obtaining an epimerization reaction product by acting on the enzyme agent, and the epimerization reaction product obtained by the production method.

Mannose is an isomer of glucose and is well known as a component sugar of the glycans present in glucomannan, hemicellulose, and cell surfaces extracted from konjac and other plants. Mannose itself is also known to be contained in cranberries.

Mannose has low bioavailability, and most of the orally ingested mannose is rapidly excreted in the urine. It is believed that the causative bacteria of urinary tract infections attach to the urinary tract surface through binding between the mannose-binding lectin possessed by the bacteria and the glycans present on the urinary tract surface cells, causing infection. Orally ingested mannose is rapidly excreted in urine, and the excreted mannose is expected to have the effect of preventing urinary tract infections by inhibiting the binding of the mannose-binding lectin of the bacteria to the glycans on the cell surface (Non-Patent Document 1).

Patent Document 1 discloses technology relating to an agent for inhibiting infection with harmful bacteria or a feed for preventing infection with harmful bacteria, which contains a mannose-containing sugar composition as an active ingredient. Patent Document 2 describes a texture improver for food and beverages, which contains mannose as an active ingredient.

There are several known methods for preparing mannose, including chemical reaction methods and enzymatic methods. Each method will be discussed below.
The first chemical reaction method involves reducing sucrose to produce sorbitol and mannitol, isolating the mannitol, and then oxidizing the mannitol to produce mannose. However, this method requires multiple steps and catalytic reactions at high temperatures and pressures, which makes it inefficient.

The second chemical reaction method is to prepare mannose by hydrolyzing glucomannan or 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 involves hydrolysis of hemicellulose such as copra meal. However, this method has the problem that the yield of mannose is poor due to the diversity of constituent sugars, and in the case of acid hydrolysis, the yield is even worse and the purification load is large.

The fourth chemical reaction method is to prepare mannose by isomerizing glucose under alkaline conditions. However, this method has problems such as low yield due to sugar decomposition and a large purification load.

The fifth chemical reaction method is a method for preparing mannose by isomerizing fructose under alkaline conditions. However, like the fourth chemical reaction method, this method has problems with yield and purification load. It also has problems with fructose being relatively expensive compared to glucose, and with fructose remaining in the reaction system, making it difficult to separate it from mannose.

The first enzymatic method is to prepare mannose by reacting fructose with mannose isomerase. However, there are problems with the cost of the raw material fructose and the separation of fructose remaining in the reaction system.

The second enzymatic method is to prepare mannose by reacting glucose or fructose with aldose-ketose isomerase (hereinafter referred to as "AKI"). However, this method also has problems with separating the fructose remaining in the reaction system.

In addition, cellobiose 2-epimerase (hereinafter referred to as "CE") is known as an enzyme that catalyzes the 2-epimeriZation (isomeriZation) reaction of the hydroxyl group at the 2nd position of a reducing end sugar residue, such as cellobiose, Which is composed of tWo or more sugar residues Where the reducing end sugar residue is bound by a β-1,4 bond, or the reverse reaction (Patent Documents 3 and 4). This enzyme can convert cellobiose, Which is bound to glucose by a β-1,4 bond, to 4-O-β-glucosylmannose, or catalyZe the reverse reaction. Patent Document 4 also describes that mannose Was obtained by acting this CE on glucose (Third Enzyme Method). However, in the third enzymatic method using CE described in Patent Document 4, CE catalyZes the aldose-ketose isomeriZation reaction in addition to the epimeriZation reaction. As a result, fructose is produced along with mannose, which creates problems when it comes to separating the by-product fructose.

The fourth enzymatic method uses an enzyme that catalyzes the epimerization reaction of the hydroxyl group at the 2-position of glucose, mannose, galactose, and talose (Patent Documents 5 and 6). This method is superior to the first to third enzymatic methods in that fructose and tagatose are hardly produced as by-products (Patent Documents 5 and 6). However, the maximum temperature at which these enzymes can stably maintain their activity is 50°C, and improvement in heat resistance is desired in consideration of their use in actual production.

Patent No. 4694667 JP 2001-275583 A Patent No. 5092049 International Publication No. 2010/090095 JP 2019-033702 A Patent No. 6657453

Sharma V, Ichikawa M, Freeze HH. Mannose metabolism: more than meets the eye. Biochem Biophys Res Commun. 2014;453(2):220‐228. doi:10.1016/j.bbrc.2014.06.021

The production efficiency of epimerization reaction products using the enzymatic method depends largely on the properties of the enzyme used. There was a demand for enzymes with higher heat resistance that are suitable for use on an industrial scale.

The problem to be solved by the present invention is to provide an enzyme agent that catalyzes an epimerization reaction that produces fewer by-products and has higher heat resistance.

The present inventors have attempted to search for an epimerization enzyme for mannose and have analyzed the expression and properties of genes encoding various proteins. In particular, expression analysis was performed focusing on a gene encoding a protein (NCBI Reference Sequence: WP_083712873.1) that is present on the genome of Paludisphaera borealis and is presumed to be an AGE family epimerase/isomerase. As a result, it was found that the protein has 2-epimerization activity that converts mannose to glucose, and galactose to talose, respectively. Furthermore, it was found that this protein has cellobiose 2-epimerase activity, as it acts on disaccharides with β-1,4 bonds, i.e., cellobiose, lactose, β-1,4-mannobiose, etc., to produce epimerization reaction products.

The inventors also discovered that the protein is stable up to 60°C, which led to the completion of the present invention.

The present invention is as follows.
[1] An enzyme preparation for catalyzing an epimerization reaction of a sugar, comprising any one of the following proteins (a) to (c), wherein the sugar is any one selected from the group consisting of glucose, mannose, talose, galactose, and disaccharides having a β-1,4 bond:
(a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 3;
(b) a protein consisting of an amino acid sequence having 90% or more amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 3 and having an activity of catalyzing an epimerization reaction of sugar;
(c) A protein having an amino acid sequence in which one or several amino acids have been substituted, inserted, deleted and/or added in the amino acid sequence set forth in SEQ ID NO: 1 or 3, and having the activity of catalyzing an epimerization reaction of sugar.
[2] An enzymatic preparation for catalyzing an epimerization reaction of a sugar, comprising a culture supernatant of a transformed cell into which any one of the following polynucleotides (d) to (f) has been introduced, wherein the sugar is any one selected from the group consisting of glucose, mannose, talose, galactose, and disaccharides having a β-1,4 bond, and having an activity of catalyzing an epimerization reaction of a sugar:
(d) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4;
(e) a polynucleotide that hybridizes under stringent conditions with a complementary strand of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4, and encodes a protein having an activity of catalyzing sugar epimerization;
(f) A polynucleotide having a base sequence identity of 90% or more to a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4, and encoding a protein having the activity of catalyzing sugar epimerization.
[3] A method for producing a sugar epimerization reaction product, comprising the step of reacting a sugar substrate with the enzyme preparation according to [1] or [2] to obtain a sugar epimerization reaction product.
[4] A method for producing a food, feed, cosmetic, or pharmaceutical, comprising a step of obtaining a food, feed, cosmetic, or pharmaceutical using an epimerization reaction product of a sugar obtained by carrying out the method according to [3].

The present invention provides a novel enzyme preparation having epimerization activity.

Figure 1A shows the amino acid sequence of a hypothetical protein from Paludysphaera borealis (SEQ ID NO: 1). The underlined region is the predicted signal peptide sequence. 1B is a nucleotide sequence (SEQ ID NO: 2) encoding the amino acid sequence of a hypothetical protein derived from Paludysphaera borealis. The underlined region is a nucleotide sequence corresponding to a signal peptide sequence. 1C is the amino acid sequence (SEQ ID NO: 3) of a hypothetical protein derived from Paludysphaera borealis. The amino acid sequence shown in SEQ ID NO: 3 is a sequence obtained by deleting a portion predicted to be a signal peptide from the amino acid sequence of SEQ ID NO: 1. 1D is a nucleotide sequence (SEQ ID NO: 4) encoding the amino acid sequence of a hypothetical protein derived from Paludysphaera borealis. The nucleotide sequence shown in SEQ ID NO: 4 is a sequence obtained by deleting the nucleotide sequence corresponding to the signal peptide sequence from the nucleotide sequence of SEQ ID NO: 2. 2 is an SDS-PAGE analysis of proteins derived from Pardisphaera borealis obtained by culturing in Example 1 (culture supernatant filtered through a filter). The band of the protein derived from Pardisphaera borealis is indicated by an arrow. 3A shows the TLC analysis of the reaction products of this enzyme. The abbreviations are as follows: Glc: glucose, Man: mannose, Gal: galactose, Tal: talose, Frc: fructose, Tag: tagatose, Xyl: xylose, B: reaction blank, R: reaction sample. Black circles (●) indicate reaction products. Figure 3B shows the TLC analysis of the reaction products of this enzyme. The abbreviations are as follows: Cel: cellobiose, Man2: β-1,4-mannobiose, Lac: lactose, Epi-Lac: epilactose, B: reaction blank, R: reaction sample. Black circles (●) indicate reaction products. 3C is a TLC analysis of the reaction product of the empty vector control. The abbreviations are as follows: Glc: glucose, Man: mannose, Gal: galactose, Tal: talose, Frc: fructose, Tag: tagatose, Xyl: xylose, B: reaction blank, R: reaction sample. 3D is a TLC analysis of the reaction products of the empty vector control. The abbreviations are as follows: Cel: cellobiose, Man2: β-1,4-mannobiose, Lac: lactose, Epi-Lac: epilactose, B: reaction blank, R: reaction sample. 4A and 4B are graphs showing the relative activity of the enzyme at each reaction pH, and the relative remaining activity of the enzyme when maintained at each pH. Figure 5A is a graph showing the relative activity of this enzyme at each reaction temperature. Black circles (●) indicate the relative activity at reaction pH 6.0, and black triangles (▲) indicate the relative activity at reaction pH 7.5. Figure 5B is a graph showing the relative remaining activity of this enzyme when maintained at each reaction temperature for 60 minutes. White circles (◯) indicate the relative remaining activity when maintained at pH 6.0, and white triangles (△) indicate the relative remaining activity when maintained at pH 7.5. 6 is a graph showing the mannose content and fructose content in the reaction solution when the present enzyme solution and the empty vector control were allowed to act on glucose for 72 hours under conditions of pH 6.0, pH 7.0, and pH 8.0, respectively. The amounts of enzyme added were 10.3, 20.6, 41.3, 82.5, 165.0, and 330.0 μL/g-ds. Black circles (●) indicate the mannose content in the reaction solution acted on by the present enzyme solution, white circles (◯) indicate the fructose content in the reaction solution acted on by the present enzyme solution, black squares (■) indicate the mannose content in the reaction solution acted on by the empty vector control, and white squares (□) indicate the fructose content in the reaction solution acted on by the empty vector control. The mannose content and fructose content were calculated from the peak area ratio of HPLC analysis. 7 is a graph showing the time course of the mannose content and fructose content in the reaction solution when the present enzyme solution and the empty vector control were reacted with glucose under conditions of pH 6.0, pH 7.0, and pH 8.0, respectively. The amount of enzyme added was 330.0 μL/g-ds. Black circles (●) indicate the mannose content in the reaction solution reacted with the present enzyme solution, white circles (◯) indicate the fructose content in the reaction solution reacted with the present enzyme solution, black squares (■) indicate the mannose content in the reaction solution reacted with the empty vector control, and white squares (□) indicate the fructose content in the reaction solution reacted with the empty vector control. The mannose content and fructose content were calculated from the peak area ratio of HPLC analysis.

The following description of the present invention may be based on representative embodiments and specific examples, but the present invention is not limited to such embodiments. In this specification, carbohydrates are referred to as D-forms unless otherwise specified. Furthermore, a numerical range expressed using "~" means a range including the numerical values before and after "~" as the lower and upper limits. In this specification, when a numerical value is accompanied by the term "about", it is intended to include a range of ±10% of that value.

The sugar composition (%) of the reaction product produced by the enzyme of the present invention was calculated as the area ratio (%) of the peak corresponding to each sugar, assuming that the total area of the peaks detected by HPLC is 100.

(Enzyme preparation)
The present invention provides an enzyme preparation for catalyzing an epimerization reaction of a sugar, comprising any one of the following proteins (a) to (c):
(a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 3;
(b) a protein consisting of an amino acid sequence having 90% or more amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 3 and having an activity of catalyzing an epimerization reaction of sugar;
(c) A protein having an amino acid sequence in which one or several amino acids have been substituted, inserted, deleted and/or added in the amino acid sequence set forth in SEQ ID NO: 1 or 3, and having the activity of catalyzing an epimerization reaction of sugar.
In this specification, any of the above proteins (a) to (c) may be referred to as the protein contained in the enzyme preparation of the present invention, the present protein, or the present enzyme.

"Epimerization of sugars" or "epimerization of sugars" refers to a reaction that inverts the configuration of one of the asymmetric carbons in a sugar.Specific examples include the epimerization (isomerization) of the hydroxyl group at the 2-position of a sugar, or the inverse reaction.

Enzymes that catalyze epimerization are usually called epimerases. For example, cellobiose 2-epimerase (CE) is one of the most widely studied epimerases. CE catalyzes the reaction of isomerizing the reducing end glucose residue of cellobiose to mannose residue.

The amino acid sequence described in SEQ ID NO:1 is an amino acid sequence obtained from the genome information of Paludisphaera borealis (NCBI Reference Sequence: WP_083712873.1). Here, although the amino acid sequence described in SEQ ID NO:1 is annotated as an AGE family epimerase/isomerase, the specific properties when expressed as a protein were not known at the time of filing of this application.

The present inventors expressed the protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 as a recombinant protein using a Bacillus subtilis expression system, and analyzed its enzyme activity, discovering that the protein acts on at least glucose, mannose, talose, galactose, cellobiose, lactose, epilactose, and β-1,4-mannobiose. That is, the present inventors discovered that the protein consisting of the amino acid sequence set forth in SEQ ID NO: 3 is an enzyme that catalyzes epimerization of the hydroxyl group on the 2-position carbon of the reducing sugar residues of monosaccharides such as glucose, mannose, talose, and galactose, as well as cellobiose, lactose, epilactose, and β-1,4-mannobiose. The amino acid sequence of SEQ ID NO: 3 is a sequence obtained by deleting the portion estimated to be a signal peptide from the amino acid sequence of SEQ ID NO: 1.

The amino acid sequence of SEQ ID NO:1 derived from Pardisphaera borealis had a primary amino acid sequence identity of 50% or less with the amino acid sequences of known CEs. Specifically, the amino acid sequence of SEQ ID NO:1 has a primary amino acid sequence identity of 37% with CE derived from Meliorivacter roseus (GenBank: AFN73749.1) (identity was evaluated using ClustalW (Larkin M. A. et al., 2007, Bioinformatics, 2007; 23:2947-2948)).

The protein contained in the enzyme preparation of the present invention may be a protein consisting of an amino acid sequence having 90% or more amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 3, and exhibiting sugar epimerization activity. In one embodiment, the protein contained in the enzyme preparation of the present invention may be a protein consisting of an amino acid sequence having 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 3, and exhibiting sugar epimerization activity.

Amino acid sequence identity is defined as the percentage of amino acid residues that are identical between two amino acid sequences after aligning the two amino acid sequences being compared and introducing gaps, if necessary, to obtain the maximum percentage sequence identity. Amino acid sequence identity can be determined, for example, by using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.

Furthermore, the protein contained in the enzyme preparation of the present invention may be a protein consisting of an amino acid sequence in which one or several amino acids have been substituted, inserted, deleted and/or added in the amino acid sequence set forth in SEQ ID NO: 1 or 3, and which has activity to catalyze an epimerization reaction of sugar. The range of "one or several" in the "amino acid sequence in which one or several amino acids have been substituted, inserted, deleted and/or added" as used herein is not particularly limited, but means, for example, about 1 to 20, preferably 1 to 10, more preferably 1 to 7, even more preferably 1 to 5, and particularly preferably 1 to 3.
For the method for producing the protein contained in the enzyme preparation of the present invention, please refer to <Production of Protein> described below.

The present invention provides an enzyme preparation for catalyzing an epimerization reaction of a sugar, comprising a culture supernatant of a transformed cell into which any one of the following polynucleotides (d) to (f) has been introduced, wherein the sugar is any one selected from the group consisting of glucose, mannose, talose, galactose, and disaccharides having a β-1,4 bond:
(d) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4;
(e) a polynucleotide that hybridizes under stringent conditions with a complementary strand of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4, and encodes a protein having an activity of catalyzing sugar epimerization;
(f) A polynucleotide having a base sequence identity of 90% or more to a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4, and encoding a protein having the activity of catalyzing sugar epimerization.

The base sequence shown in SEQ ID NO:2 is a sequence that encodes the amino acid sequence of SEQ ID NO:1 (NCBI Reference Sequence: WP_083712873.1) obtained from the genome information of Paludisphaera borealis. The base sequence shown in SEQ ID NO:4 is a sequence obtained by deleting the base sequence encoding the portion predicted to be a signal peptide from the base sequence of SEQ ID NO:2.

The present invention also includes a culture supernatant of a transformed cell into which a polynucleotide that hybridizes under stringent conditions with a complementary strand of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4 and that encodes a protein having activity of catalyzing an epimerization reaction has been introduced. The term "polynucleotide that hybridizes under stringent conditions with a complementary strand of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4" refers to a polynucleotide (e.g., DNA) that can be obtained by employing a colony hybridization method, a plaque hybridization method, a Southern blot hybridization method, or the like, using the complementary strand of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4 as a probe. An example of "stringent conditions" is a DNA that can be identified by hybridization at 65°C in the presence of 0.7 to 1.0 M sodium chloride, followed by washing the filter at 65°C using a 0.1 to 5x SSC solution (1x SSC composition: 150 mM sodium chloride, 15 mM sodium citrate) (Molecular Cloning: A Laboratory Manual ^2nd Ed (Sambrook, Maniatis et al., Cold Spring Harbor Laboratory Press (1989))).

Also included in the present invention is a culture supernatant of a transformed cell into which a polynucleotide encoding a protein having an activity of catalyzing an epimerization reaction has been introduced, the polynucleotide having a base sequence identity of 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with respect to a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4. Base sequence identity is defined as the percentage of bases that are identical between two base sequences after aligning the two base sequences to be compared and introducing gaps, if necessary, to obtain the maximum percent sequence identity. Base sequence identity can be determined by using publicly available computer software such as, for example, BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software.

The transformed cells are preferably Bacillus subtilis. The culture supernatant of the present invention is substantially free of cells, and the cells are removed by filtration or centrifugation.

The culture supernatant of the present invention contains a protein which is an expression product of the polynucleotides (d) to (f) above. In this specification, the protein which is an expression product of the polynucleotides (d) to (f) above and which is contained in the culture supernatant of the present invention may be referred to as the protein contained in the enzyme preparation of the present invention, the present protein, or the present enzyme.

The epimerization activity of the protein contained in the enzyme preparation of the present invention is evaluated by allowing the protein to act on a substrate selected from glucose, mannose, talose, galactose, cellobiose, β-1,4-mannobiose, lactose, and epilactose, and detecting the epimerization reaction product obtained.

The reaction products can be detected using high performance liquid chromatography (HPLC) with an RI detector or a charged aerosol detector. Detection can also be performed using thin layer chromatography (TLC) and high performance ion exchange chromatography with pulsed amperometric detection (HPAEC-PAD).

Epimerization of glucose produces mannose, epimerization of mannose produces glucose, epimerization of talose produces galactose, epimerization of galactose produces talose, epimerization of lactose produces epilactose, epimerization of epilactose produces lactose, epimerization of cellobiose produces 4-O-β-glucosyl-mannose, and β-1,4-mannobiose produces 4-O-β-mannosyl-glucose.

The enzyme preparation for catalyzing the epimerization reaction of sugars of the present invention can be used to catalyze the epimerization of a hydroxyl group bonded to the carbon at the 2-position of a reducing sugar residue of a monosaccharide or disaccharide. In one embodiment of the present invention, the enzyme preparation for catalyzing the epimerization reaction of sugars of the present invention can be used to catalyze the epimerization reaction of any sugar selected from, but not limited to, at least glucose, mannose, talose, galactose, cellobiose, β-1,4-mannobiose, lactose and epilactose. This is because the protein contained in the enzyme preparation of the present invention acts on at least glucose, mannose, talose, galactose, cellobiose, β-1,4-mannobiose, lactose and epilactose. The protein contained in the enzyme preparation of the present invention can epimerize the hydroxyl group at the 2-position of a monosaccharide and the hydroxyl group at the 2-position of the reducing end of a disaccharide having a β-1,4 bond.

The enzyme preparation for catalyzing the epimerization reaction of sugars of the present invention can be used to produce a sugar epimerization reaction product. Specifically, the enzyme preparation for catalyzing the epimerization reaction of sugars of the present invention can be used to produce mannose using glucose as a substrate, and can also be used to produce glucose using mannose as a substrate. In one embodiment of the present invention, the enzyme preparation is preferably used to produce mannose using glucose as a substrate. This is because glucose is industrially mass-produced and inexpensive, and can therefore be easily procured to produce mannose, which has problems with industrial production methods.

The enzyme preparation for catalyzing the epimerization reaction of sugars of the present invention can be used to produce talose using galactose as a substrate, and can also be used to produce galactose using talose as a substrate. In one embodiment of the present invention, the enzyme preparation is preferably used to produce talose using galactose as a substrate. This is because galactose can be obtained by hydrolysis of lactose, which is abundant in nature, and can therefore be easily procured for producing talose, which is a rare sugar.

The enzyme agent for catalyzing the epimerization reaction of sugars of the present invention may or may not substantially use N-acylglucosamine as a substrate, but preferably does not substantially use it as a substrate. In this specification, "substantially does not use a specific sugar as a substrate" means, for example, that when the activity using mannose as a substrate is taken as 100%, the relative activity using the specific sugar as a substrate is 5% or less, preferably 1% or less, more preferably 0%, and even more preferably, the activity using the specific sugar as a substrate is not measurable, and particularly preferably, the catalytic reaction using the specific sugar as a substrate does not occur at all.

The protein contained in the enzyme preparation of the present invention has a molecular weight measured by SDS-PAGE in the range of 45-55 kDa or approximately 48 kDa. The molecular weight of the protein shown here is the molecular weight of the protein expressed by inserting DNA encoding the protein into a plasmid and using Bacillus subtilis as a host cell. The theoretical value of the molecular weight of the protein measured in Examples 1 and 2 as being in the range of 45-55 kDa or approximately 48 kDa is 48.1 kDa.

The proteins contained in the enzyme preparation of the present invention can act on, but are not limited to, at least glucose, mannose, talose, galactose, cellobiose, β-1,4-mannobiose, lactose and epilactose, and can catalyze the epimerization reaction of these sugars.

Some conventional cellobiose 2-epimerases produce mannose and fructose from glucose (Patent Document 3, Patent Document 4). However, the protein contained in the enzyme preparation of the present invention can convert glucose mainly to mannose. In a reaction system using this protein, a small amount of fructose may be produced from glucose, but it is only a trace amount.

When measured at 37°C using mannose as a substrate, this protein exhibits maximum activity at pH 7.5, and the optimum pH at which it exhibits 80% or more of the maximum activity is 7.3 to 7.8. In a test in which the protein was kept at 4°C for 24 hours, this protein exhibited 80% or more of residual activity at pH 4.6 to 8.4 and was stable. The optimum pH and pH stability were measured by measuring the amount of glucose produced under the conditions shown in Examples 3-1 and 3-2.

When measured at pH 7.5 using mannose as a substrate, this protein exhibits maximum activity at a temperature of 50°C, and the optimum temperature range for activity of 80% or more of the maximum activity is 40°C to 55°C. When measured at pH 6.0, this protein also showed a tendency for activity to be high at temperatures of 60°C and 65°C. In a test in which this protein was maintained at temperatures of 30°C to 70°C for 60 minutes, this enzyme was stable at pH 7.5, exhibiting a residual activity of 90% or more at 50°C or less, and was stable at pH 6.0, exhibiting a residual activity of 90% or more at 60°C or less. The optimum temperature and temperature stability were measured by measuring the amount of glucose produced under the conditions shown in Examples 3-3 and 3-4.

The enzyme preparation of the present invention may contain, in addition to the above-mentioned protein having activity to catalyze the epimerization reaction of sugar, further components as long as they do not inhibit the epimerization reaction of sugar. These may be components used in ordinary enzyme preparations, such as buffers, stabilizers, excipients, etc. Such further components are known from the prior art and are well known to those skilled in the art. The enzyme preparation of the present invention is also not particularly limited in shape, and may be a solid (e.g., powder) or liquid. The enzyme preparation of the present invention can be used, for example, by adding a solid or liquid to a solution of a sugar substrate.

The enzyme preparation of the present invention can be provided, for example, as an enzyme preparation in which a protein having activity to catalyze the sugar epimerization reaction of the present invention is immobilized on an immobilization carrier. By using the immobilized enzyme preparation, for example, it is possible to produce a reaction product at a high substrate concentration and at a high temperature, and it is also possible to produce a reaction product using a bioreactor system.

The above-mentioned immobilization carrier is not particularly limited, and any carrier can be used as long as it can adsorb or crosslink a protein having activity to catalyze the epimerization reaction of sugars and can retain the activity of the protein.

Examples of the immobilization carrier include an anion exchange carrier, a cation exchange carrier, and a hydrophobic carrier. A specific example of the immobilization carrier is an ion exchange gel. Examples of the ion exchange gel include the Diaion (registered trademark) series (manufactured by Mitsubishi Chemical Corporation) such as Diaion (registered trademark) SK1B, Diaion (registered trademark) PK212, Diaion (registered trademark) HPA25, Diaion (registered trademark) UBK550, and UBK555; the Sepabeads (registered trademark) series (manufactured by Mitsubishi Chemical Corporation) such as Sepabeads (registered trademark) SP-207 and Sepabeads (registered trademark) SP-850; and the Duolite series (manufactured by Sumika Chemtex Corporation) such as Duolite A568, Duolite PWA7, and Duolite XAD761.

The enzyme preparation of the present invention can be used for sugars other than glucose, mannose, talose, galactose, cellobiose, β-1,4-mannobiose, lactose and epilactose as long as the protein contained in the enzyme preparation of the present invention can catalyze epimerization.
The shape of the immobilization carrier is not particularly limited, and examples thereof include membranes, beads, plates, and the like.

The method for immobilizing a protein having activity to catalyze the epimerization reaction of sugars to the above-mentioned immobilization carrier is not particularly limited, and can be carried out, for example, by adding the protein and the above-mentioned immobilization carrier to a solvent such as a buffer solution and shaking the mixture.

(Method for producing epimerization reaction products of sugars)
The present invention provides a method for producing a sugar epimerization reaction product, comprising the step of reacting a sugar substrate with an enzyme agent for catalyzing a sugar epimerization reaction of the present invention to obtain a sugar epimerization reaction product.

The enzyme preparation of the present invention can be applied to a sugar substrate by, for example, preparing an aqueous solution of the sugar substrate, adjusting the pH as necessary, and then adding the enzyme preparation of the present invention to the aqueous solution. Furthermore, the enzyme preparation can be prepared by immobilizing a protein having the activity of catalyzing the sugar epimerization reaction of the present invention on a carrier, and contacting the enzyme preparation with an aqueous solution of the sugar substrate. This is because the sugar epimerization reaction using the enzyme preparation of the present invention can be carried out in the reaction solution.

The sugar substrate that can be used in the method for producing an epimerization reaction product of sugar may be any one selected from glucose, mannose, talose, galactose, cellobiose, β-1,4-mannobiose, lactose and epilactose, but is not limited thereto. Any other sugar can be used as the sugar substrate as long as the protein contained in the enzyme preparation of the present invention can catalyze epimerization. As the sugar substrate, a pure product may be used, but a sugar composition containing other carbohydrates may also be used from the viewpoint of cost, etc. Specifically, for example, when glucose is used as the main substrate, the sugar composition may contain, in addition to glucose, maltose, maltotriose, and the like that are generated by the decomposition of starch.

There are no particular limitations on the reaction temperature when the protein contained in the enzyme preparation of the present invention is allowed to act on the sugar substrate, so long as it is within the temperature range in which the enzyme activity is expressed. As described in the Examples, the protein contained in the enzyme preparation of the present invention can react at a temperature of 65°C or lower when reacted at pH 6.0. Some conventional epimerases have limitations on their use on an industrial scale due to their thermal stability. However, the protein contained in the enzyme preparation of the present invention exhibits high heat resistance and has thermal stability suitable for use on an industrial scale.

Therefore, the reaction temperature (liquid temperature of the reaction solution) when the enzyme preparation of the present invention is applied can be set in a wide temperature range, such as 35 to 65°C. Accurate temperature control can be difficult in industrial-scale reaction systems, but this protein has the advantage that it can be used stably over a wide temperature range.

The reaction temperature can be set in the range of 35 to 65°C, but in the case of pH 6.0, it is preferable to set it in the range of 50 to 60°C. If the reaction temperature is 50°C or higher, contaminating bacteria that use sugar as a nutrient are less likely to grow, and at 60°C or lower, the protein is stable.

In the method for producing an epimerization reaction product of sugar, the enzyme preparation of the present invention can be used in combination with other enzymes, as described below. When other enzymes are used in combination, the reaction temperature may be within a temperature range in which the protein contained in the enzyme preparation of the present invention acts stably. As described above, since the protein contained in the enzyme preparation of the present invention is stable over a wide temperature range, it is often possible to set the temperature within a range in which the activity of all enzymes used in the reaction system can be fully utilized, taking into account the temperature at which the other enzymes used in combination are stably activated.

The reaction temperature does not need to be constant throughout the entire reaction time. If it is desired to increase the activity of another enzyme used in combination in the early part of the reaction time, the temperature at the early stage of the reaction can be set to a temperature range in which the activity of that enzyme is high, and in the middle and late stages of the reaction time, the temperature can be set to a temperature range in which the activity of the protein contained in the enzyme preparation of the present invention is high. It can be adjusted as appropriate.

The pH of the reaction solution when the protein contained in the enzyme preparation of the present invention is allowed to act on the sugar substrate is not particularly limited as long as it is within the pH range in which the enzyme activity is expressed, but can be set in the range of pH 5.5 to 8.5. This is because the protein contained in the enzyme preparation of the present invention is capable of reacting at least in the range of pH 4.0 to 8.5, as described in the Examples. In order to efficiently obtain a sugar epimerization reaction product, it is preferable to set the pH of the reaction solution when the protein contained in the enzyme preparation of the present invention is allowed to act in the range of pH 6.0 to 8.0. The pH of the reaction solution can be adjusted by adding an acid or alkali as necessary.

In the method for producing an epimerization reaction product of sugar, the reaction time of the substrate and the protein contained in the enzyme preparation of the present invention can be appropriately determined taking into consideration the reaction temperature, the concentration of the substrate, and, when other enzymes are used in combination, the characteristics of the enzymes used. In addition, based on the conventional technology in this field, a suitable reaction time for efficiently producing an epimerization reaction product can be appropriately determined. Specific examples of reaction times include 10 minutes to 72 hours, but are not intended to be limited to these. During the reaction, the enzyme preparation of the present invention can be added as appropriate.

The substrate concentration in the reaction solution is not particularly limited, and for example, within the range in which the enzyme reaction can proceed, the higher the substrate concentration, the more economically advantageous it is. When glucose is used as the substrate, the glucose concentration is in the range of 0.1 g to 300 g per 100 g of solvent. The solvent of the reaction solution is not particularly limited, and examples thereof include water, buffer solutions, and the like. In addition, when the solubility of the selected substrate in the solvent is low, for example, the substrate may not be completely dissolved at the desired concentration. When the enzyme reaction is performed in a batch system, for example, the substrate does not need to be completely dissolved at the beginning of the reaction, and a substrate concentration that is dissolved at the end of the enzyme reaction may be selected. On the other hand, when a column filled with an immobilized enzyme agent is used in the enzyme reaction, for example, the substrate is preferably completely dissolved in order to avoid pressure loss due to clogging of the column. In addition, when a column filled with an immobilized enzyme agent is used in the enzyme reaction, for example, it is preferable to reflux the substrate solution through the column and pass it continuously. In addition, when using the enzyme preparation of the present invention to produce mannose from glucose as a substrate, starch syrup, powdered syrup, hydrol (molasses liquid produced during the production of crystalline glucose), etc. can be used. Furthermore, for example, carbohydrates containing glucose as a constituent sugar, such as dextrin or starch, can be used as a raw material, and hydrolases can be added together with the enzyme preparation of the present invention, so that mannose can be produced from glucose while producing the substrate glucose from the raw material.

As a specific example of the production of an epimerization reaction product using the enzyme preparation of the present invention, when glucose is used as a substrate, an aqueous glucose solution having a solids concentration of about 1 to 65% is prepared, the pH of the aqueous solution is adjusted to about 6.0 to 8.0 using an acid or alkali as necessary, the enzyme preparation of the present invention is added to the aqueous solution, and the solution is maintained at a temperature in the range of 35 to 65°C for about 1 to 72 hours to produce mannose.

In addition, in the method for producing an epimerization reaction product of sugar, the enzyme preparation of the present invention can be used in combination with other enzymes. The other enzymes are not limited to those, and may be those that produce sugars that can be substrates for the enzymes of the present invention that have the activity of catalyzing the epimerization reaction of sugar. For example, an enzyme that hydrolyzes carbohydrates that contain galactose as a constituent sugar, such as lactose, can be used as the other enzyme. By using the enzyme preparation of the present invention (i.e., an enzyme preparation for catalyzing the epimerization reaction of sugar) in combination with the hydrolase as the other enzyme, galactose can be produced by hydrolysis from carbohydrates that contain galactose as a constituent sugar, such as lactose, while talose can be produced from galactose by epimerization.

The above production method produces an aqueous sugar solution containing an epimerization reaction product. This aqueous sugar solution containing the epimerization reaction product can be decolorized, desalted, purified, etc., as necessary, using conventional methods. In addition, the aqueous sugar solution containing the epimerization reaction product can be subjected to resin fractionation, precipitation with an organic solvent such as ethanol, chromatographic fractionation, ultrafiltration membrane treatment, etc., to remove unreacted substrates and increase the purity of the epimerization reaction product. The purification process can be performed by a single operation or by combining several operations to purify the epimerization reaction product more efficiently.

In another embodiment, the present invention provides a method for producing a sugar epimerization reaction product, comprising the step of treating a sugar substrate with an enzyme agent for catalyzing the sugar epimerization reaction of the present invention under conditions of pH 5.5 to 7.0 to obtain a sugar epimerization reaction product, in which by-products in the reaction product are suppressed to 5% or less of the target epimerization reaction product. For example, mannose can be produced by treating glucose with an enzyme agent for catalyzing the sugar epimerization reaction of the present invention under conditions of pH 6.0 to 7.0, and in this method, the by-products of fructose in the reaction product can be suppressed to 5% or less of mannose. The pH conditions are preferably in the range of pH 6.0 to 6.5. The by-products in the reaction product are preferably suppressed to 4.5% or less, 4.0% or less, 3.5% or less, 3.0% or less, 2.5% or less, 2.0% or less, 1.5% or less, 1.0% or less, or 0.5% or less of the target epimerization reaction product.

The present invention provides a method for producing a food, feed, cosmetic, or pharmaceutical, which includes a step of obtaining a food, feed, cosmetic, or pharmaceutical using a sugar epimerization reaction product obtained by carrying out a method for producing a sugar epimerization reaction product.

The sugar epimerization reaction product can be obtained by carrying out the above-mentioned method for producing a sugar epimerization reaction product.

In the process of obtaining food, feed, cosmetic, or pharmaceutical using the epimerization reaction product of sugar obtained by carrying out the method for producing an epimerization reaction product of sugar, the epimerization reaction product can be used as one of the raw materials to produce the food, feed, cosmetic, or pharmaceutical, or the epimerization reaction product itself can be prepared in an appropriate form (powder, liquid, etc.) and provided as the food, feed, cosmetic, or pharmaceutical.

Examples of foods that can be produced by the method of the present invention include, but are not limited to, various carbohydrates (bread, noodles, cooked rice, mochi), various Japanese sweets (rice crackers, arare, okoshi, gyuhi, mochi, manju, dorayaki, uiro, bean paste, yokan, mizuyokan, kingyoku, castella, candy), various Western sweets (bread, biscuits, crackers, cookies, pies, donuts, steamed cakes, puddings, jellies, mousses, bavarois, custard cream, cream puffs, waffles, sponge cakes, chocolates, chewing gums, caramels, nougat, candies, syrups), various frozen desserts (ice cream, sorbets, gelato, shaved ice), various paste-like foods (flower paste, peanut paste, margarine, fruit paste), various beverages (fruit juice-containing beverages, fruit juice, vegetable juice, cider, ginger ale, isotonic beverages, amino acid beverages, jelly beverages, coffee beverages, green tea, black tea, oolong tea, barley tea, milk beverages, lactic acid bacteria beverages, cocoa, beer, happoshu, third beer, non-alcoholic beverages, beer-flavored beverages, liqueurs, chuhai, sake, fruit wine, distilled alcohol, nutritional drinks, health beverages, powdered beverages), processed fruit and vegetable products (jams, marmalades, preserved in syrup, candied fruit, pickles), various dairy products (cheese, yogurt, butter, condensed milk, powdered milk), powdered foods (powdered soup, powdered mousse, powdered jelly, powdered sweetener), nutritional foods, diet foods, nutritional foods for sports, liquid foods, semi-solid liquid foods, nursing care foods, foods for dysphagia, etc.

Examples of feed and bait produced by the method of the present invention include, but are not limited to, feed and bait for livestock, poultry, seafood, and insects (honeybees, silkworms, etc.). These may be in the form of powder, pellets, tablets, paste, capsules, etc.

Examples of cosmetics produced by the method of the present invention include, but are not limited to, moisturizers and beauty agents. These may take the form of lotions, creams, emulsions, etc.

Examples of pharmaceuticals produced by the method of the present invention include, but are not limited to, anti-obesity agents and blood glucose level rise inhibitors, and may be in the form of tablets, powders, liquids, capsules, etc.

<Production of Protein>
The method for obtaining the protein contained in the enzyme preparation of the present invention is not particularly limited, and the protein may be a protein synthesized by chemical synthesis, or a recombinant protein produced by gene recombination technology. The case of producing a recombinant protein will be described below.

Proteins having the amino acid sequence shown in SEQ ID NO: 1 or 3, or an amino acid sequence having 90% or more amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 3, and an amino acid sequence in which one or several amino acids have been substituted, inserted, deleted and/or added in the amino acid sequence described in SEQ ID NO: 1 or 3 can be prepared by genetic engineering techniques. For example, a gene encoding the amino acid sequence of SEQ ID NO: 1 or 3 can be replicated in a host cell, or can be incorporated into a chromosome and expressed as a DNA molecule containing the gene, particularly inserted into an expression vector, and the host cell can be transformed to produce a protein by culturing the host cell. This DNA molecule can be obtained by incorporating a DNA fragment encoding the amino acid sequence shown in SEQ ID NO: 1 or 3, an amino acid sequence having 90% or more amino acid sequence identity to the amino acid sequence shown in SEQ ID NO: 1 or 3, or an amino acid sequence in which one or several amino acids have been substituted, inserted, deleted and/or added in the amino acid sequence described in SEQ ID NO: 1 or 3, into a vector molecule. According to a preferred embodiment of the present invention, the vector is a plasmid. The DNA molecule of the present invention can be produced according to the method described in Molecular Cloning: A Laboratory Manual.

The vectors that can be used in the present invention can be appropriately selected from viruses, plasmids, cosmid vectors, etc., taking into consideration the type of host cell used. For example, when the host cell is Bacillus subtilis, the pJEXOPT2 series (see Patent No. 5,126,879) and pHT series plasmids can be used; when the host cell is E. coli, λ phage series bacteriophages, pET series, pUC series, pCold series, and pGEX series plasmids can be used; when the host cell is yeast, the YEp, YCp series, and YIp series vectors, or pLeu4, pPPLeu4, and pJPLeu4 series (described in JP Patent Publication 4-218382), but are not limited to these. This plasmid may contain a marker for selecting transformants, and the selection marker can be, but is not limited to, a drug resistance marker or an auxotrophic marker gene.

Furthermore, the expression vector that can be used in the present invention can have a DNA sequence necessary for the expression of an enzyme gene, such as a promoter, a terminator, a ribosome binding site, a transcriptional regulatory signal such as a transcription termination signal, a translational regulatory signal, etc. As the promoter, a promoter such as subtilisin or SPAC can be used in Bacillus subtilis, and a promoter such as alcohol dehydrogenase (ADH), acid phosphatase (PHO), galactose gene (GAL), or glyceraldehyde-3-phosphate dehydrogenase gene (GAP) can be used in yeast, but is not limited to these. The addition of a signal peptide is preferable because it has the advantage that the target enzyme is secreted into the culture supernatant, making purification easier. In addition, the signal peptide can be replaced with one derived from Bacillus subtilis or yeast (for example, an invertase signal, an acid phosphatase signal, a λ-factor signal, etc.). In addition, in E. coli, in addition to the commonly used lac promoter and T7 promoter, it is possible to make the expression more efficient by using a cspA promoter or the like to simultaneously express a molecular chaperone.

Transformed host cells can be cultured using methods commonly used for the host cells used. Usually, the enzyme is produced and accumulated in the intracellular or extracellular culture after 1-4 days of culture. Culture conditions (medium, pH, temperature, etc.) are typically 25-37°C for bacteria, 25-30°C for yeast, and about 37°C for eukaryotic cells. For information on culture conditions, see the Gene Expression Experiment Manual (Kodansha), etc.

As host cells, bacteria such as Escherichia coli and Bacillus subtilis, yeasts such as Candida utilis, Saccharomyces cerevisiae, and Pichia pastoris, as well as Rhizopus niveus, Rhizopus delmar, and higher eukaryotes (e.g., CHO cells) can be used. Bacillus subtilis is known to secrete proteins outside the bacterial cell, and some strains secrete almost no protease, and it is also preferable to use such strains as hosts. In the present invention, the host cell is preferably a yeast, a filamentous fungus, or a bacterium, more preferably a bacterium, and particularly preferably Escherichia coli or Bacillus subtilis.

The isolation and purification of proteins produced by transformants can be carried out by appropriately combining known separation and purification methods. Examples of these separation and purification methods include methods that utilize differences in solubility, such as salt precipitation and solvent precipitation; methods that utilize differences in molecular weight, such as dialysis, ultrafiltration, gel filtration, and SDS-polyacrylamide electrophoresis; methods that utilize differences in charge, such as ion exchange chromatography; methods that utilize differences in hydrophobicity, such as hydrophobic chromatography and reverse phase chromatography; and methods that utilize differences in isoelectric point, such as isoelectric focusing. In addition to the purification methods described in the examples, reference can be made to Basic Experimental Methods for Proteins and Enzymes (Nankodo) for general separation and purification methods.

The present invention will be described in more detail based on the following examples, but the present invention is not limited to these examples. In this specification, unless otherwise specified, "%" is based on mass, and the numerical range is described as including its end points. In addition, unless otherwise specified, the operating procedure was performed according to the method described in Molecular Cloning: A Laboratory Manual (Sambrook, Maniatis et al., Cold Spring Harbour Laboratory Press (1989)).

In this example, a gene encoding a protein with unidentified function (NCBI Reference Sequence: WP_083712873.1) presumed to be an AGE family epimerase/isomerase was cloned from Paludisphaera borealis to obtain a recombinant protein, and the enzyme properties were evaluated.

Example 1: Extracellular expression of the present enzyme using Bacillus subtilis as a host 1-1. Construction of expression plasmid Various microorganisms were searched for novel enzymes, and an amino acid sequence derived from Paludisphaera borealis (NCBI Reference Sequence: WP_083712873.1) was selected and functionally analyzed. This amino acid sequence (SEQ ID NO: 1) and the base sequence of the gene encoding the amino acid sequence (SEQ ID NO: 2) are shown in Figures 1A and 1B, respectively. The amino acid sequence of SEQ ID NO: 3 shown in Figure 1C is a sequence obtained by deleting a portion estimated to be a signal peptide from the amino acid sequence of SEQ ID NO: 1. The base sequence of SEQ ID NO: 4 shown in Figure 1D is a sequence obtained by deleting a base sequence corresponding to the signal peptide sequence of SEQ ID NO: 2. (Hereinafter, SEQ ID NO: 4 is referred to as the target gene.) The base sequences shown in SEQ ID NOs: 2 and 4 have been codon-optimized in order to achieve good expression in Bacillus subtilis.

An expression plasmid was constructed for expressing a target gene in Bacillus subtilis. First, a plasmid in which the base sequence described in SEQ ID NO: 2 was inserted into a pUC57 vector was used as a template, and the target gene was amplified by PCR using a primer to which a base sequence homologous to the end of the signal peptide sequence of the vector was added for sense strand amplification, and a primer to which a part of the terminator sequence was added for antisense strand amplification. The composition of the reaction solution for PCR amplification is shown below.
2x Primestar Max Premix (Takara Bio) 50μL
10 μM primer (PbAGE-Fw) 2 μL
10 μM primer (PbAGE-Rv) 2 μL
1ng/μL template 2μL
_H2O 44μL

The primers used are shown in Table 1.

The PCR amplification reaction program was as follows: first, hold at 96° C. for 1 minute, then perform 35 cycles of 98° C. for 10 seconds, 55° C. for 5 seconds, and 72° C. for 10 seconds, and then hold at 72° C. for another 5 minutes. The PCR product obtained was subjected to agarose gel electrophoresis, and the band corresponding to the amplified fragment (1305 bp) was excised from the gel and extracted and purified using illustra ^™ GFX ^™ PCR DNA and Gel Band Purification Kit (GE Healthcare).

To prepare a linearized plasmid for use in the In-Fusion (registered trademark) cloning reaction, PCR was performed using the primers shown in Table 2 and a plasmid modified from the vector pJEXOPT2 (see Patent No. 5,126,879) for optimization in this example as a template. For amplification of the antisense strand, a primer designed to amplify from the end of the signal peptide sequence of the vector was used.

The primers used are shown in Table 2.

The composition of the reaction solution for PCR amplification was the same as that used for amplifying the target gene, except for the primers and template. The PCR amplification reaction program was first held at 96 ° C for 1 minute, followed by 35 cycles of 98 ° C for 10 seconds → 55 ° C for 5 seconds → 72 ° C for 35 seconds, and then held at 72 ° C for 5 minutes. The obtained PCR product was subjected to agarose gel electrophoresis, and the band corresponding to the amplified fragment (6,935 bp) was excised from the gel, extracted and purified using illustra ^™ GFX ^™ PCR DNA and Gel Band Purification Kit (GE Healthcare). The amplified DNA fragment of the target gene and the amplified fragment of the vector pJEXOPT2 were ligated using In-Fusion HD Cloning Kit (Takara Bio). The ligation reaction was performed by holding at 50 ° C for 15 minutes.

E. coli DH5α was transformed with 2.5 μL of the ligation reaction solution, and plasmid DNA was prepared from the culture medium using illustra ^™ plasmidPrep Mini Spin Kit (GE Healthcare). The obtained plasmid was designated as the “plasmid for expressing the present enzyme.”

1-2. Expression of recombinant protein The above-mentioned plasmid for expressing the present enzyme was introduced into protoplasted Bacillus subtilis ISW1214 (Takara Bio) and cultured for 2 days at 30°C on DM3 regenerated agar medium (composition: 8.1% sodium succinate, 1% agar, 0.5% casamino acid, 0.5% yeast extract, 0.15% potassium dihydrogen phosphate, 0.35% potassium dihydrogen phosphate, 0.5% glucose, 0.4% magnesium chloride, 0.01% bovine serum albumin, 0.001% methionine, and 0.001% leucine) containing 7.5 μg/mL tetracycline. The resulting colonies were used for preculture, and then cultured for 72 hours (culture was performed as described in Japanese Patent No. 5126879, but the composition of the medium was modified). The mixture was centrifuged (5,000×g, 4° C., 5 min), and the supernatant was filtered through a 0.45 μm pore size filter (Merck) to obtain the enzyme solution. The enzyme solution was subjected to SDS-PAGE, and it was confirmed that a band appeared near the target size (48,150) (FIG. 2).

Comparative Example 1: Preparation of an empty vector control (negative control) As a negative control, a culture supernatant of cells transformed with an empty vector was prepared. A plasmid modified from the vector pJEXOPT2 (see Patent No. 5126879) for optimization in this example was introduced into protoplasted Bacillus subtilis ISW1214 (Takara Bio) and cultured at 30°C for 2 days on a regenerated agar medium (composition: 8.1% sodium succinate, 1% agar, 0.5% casamino acid, 0.5% yeast extract, 0.15% potassium dihydrogen phosphate, 0.35% potassium dihydrogen phosphate, 0.5% glucose, 0.4% magnesium chloride, 0.01% bovine serum albumin, 0.001% methionine, and 0.001% leucine) containing 7.5 μg/mL tetracycline. The colonies obtained were used for pre-culture and main culture in the same manner as in Example 1-2. The resulting culture medium was centrifuged (5,000×g, 4° C., 5 minutes), and the supernatant was filtered through a filter with a pore size of 0.45 μm (Merck) to prepare an empty vector control (negative control).

Example 2: TLC analysis of enzyme reaction products In this example, the substrates on which the enzyme acts were examined. The substrates used in the enzyme reaction were glucose, mannose, galactose, talose, fructose, xylose, cellobiose, β-1,4-mannobiose, lactose, and epilactose.
In this example, the enzyme solution was dialyzed against 20 mM MES-NaOH buffer (pH 6.5) containing 50 mM NaCl using Amicon (10K). 50 μL of reaction solution consisting of 5 μL of 100 mM HEPES-NaOH buffer (pH 7.0), 25 μL of 100 mM various substrates, 5 μL of the enzyme solution or inactivated enzyme solution (boiled for 10 minutes), and 15 μL of ultrapure water was kept at 37° C. for 1 day, and 0.5 μL of this solution was subjected to thin layer chromatography (TLC). The same reaction procedure was also performed for the empty vector control.

A different developing solvent was used for each substrate. When a monosaccharide was used as the substrate, a mixture of 2-propanol/1-butanol/water (mixture ratio of 2:2:1 (all by volume)) was used, and when a disaccharide was used as the substrate, a mixture of 2-propanol/1-butanol/water (mixture ratio of 12:3:4 (all by volume)) was used.
Sugar was detected by spraying a mixture of anisaldehyde/sulfuric acid/acetic acid (mixture ratio of 1:2:97 (all by volume)) as a coloring agent onto a TLC plate and then heating it.

The results of this enzyme are shown in Figures 3A and 3B. Differences were observed in the migration distances of sugars between the reaction blank (reaction solution to which the inactivated enzyme was added) and the reaction sample (reaction solution to which this enzyme was added) for glucose, mannose, galactose, talose, cellobiose, β-1,4-mannobiose, lactose, and epilactose. In the analysis using glucose and mannose as substrates, spots corresponding to the mobility of mannose and glucose were observed in the sample after the reaction. In the analysis using galactose and talose as substrates, spots corresponding to the mobility of talose and galactose were observed in the sample after the reaction. In the analysis using lactose and epilactose as substrates, spots corresponding to the mobility of epilactose and lactose were observed in the sample after the reaction. It was revealed that this enzyme derived from Pardisphaera borealis acts on at least glucose, mannose, galactose, talose, cellobiose, β-1,4-mannobiose, lactose, and epilactose.
The results of the empty vector control are shown in Figures 3C and 3D. For any substrate, no difference was observed in the migration distance of sugar between the reaction blank and the reaction sample, indicating that the action of this enzyme solution on the above substrates was due to the protein derived from Pardisphaella borealis, not due to contaminating enzymes.

Example 3: Properties of the enzyme (pH and temperature)
To investigate the pH and temperature characteristics of this enzyme, the activity on mannose was measured based on the amount of glucose produced as follows: 1 U of enzyme activity unit was defined as the amount of enzyme that produces 1 nmol of glucose per minute of each reaction.

3-1. Optimum pH
200 μL of reaction solution consisting of 40 μL of 500 mM mannose, 80 μL of 100 mM Britton-Robinson buffer (pH 4.0-9.5), and 80 μL of the enzyme solution diluted appropriately was kept at 37°C for 60 minutes. 20 mM MES-NaOH buffer (pH 6.5) containing 50 mM NaCl was used as the dilution solution. After 60 minutes of reaction, 40 μL of 2.0 M HCl was added, and the reaction was stopped by keeping at 100°C for 5 minutes. 40 μL of 2.0 M NaOH was added to neutralize the mixture, and centrifuged at 16,500×g and 4°C for 5 minutes. 140 μL of the supernatant after centrifugation was mixed with 60 μL of a color-developing reagent (154 mM Tris-HCl, 15.4 mM MgCl ₂ , 16.7 mM Thio-NAD+ (Oriental Yeast), 23.3 mM ATP (Wako Pure Chemical Industries, Ltd.), 6.7 U/mL hexokinase (GRADE-I, Oriental Yeast), and 6.7 U/mL glucose-6-phosphate dehydrokinase (Toyobo)) and incubated at 37° C. for 20 minutes. After incubation, the absorbance at 405 nm was measured to determine the amount of glucose produced by the reaction. A mixed solution containing 100 mM mannose and 0 to 4 mM glucose was used to create a calibration curve.

The results are shown in Figure 4A. When mannose was used as a substrate, this enzyme had maximum enzymatic activity at pH 7.5. In addition, activity of 80% or more of the maximum activity was observed at pH 7.3 to 7.8. In Figure 4, activity at pH 7.5 was set to 100%, and activity at each reaction pH was shown as a relative value.

3-2. pH stability 50 μL of a solution consisting of 40 μL of 50 mM Britton-Robinson buffer (pH 4.5-10.0) and 10 μL of the present enzyme solution was kept at 4° C. for 1 day, and the residual activity was determined according to the following method. 200 μL of a reaction solution consisting of 40 μL of 500 mM mannose, 80 μL of 200 mM HEPES-NaOH buffer (pH 7.5), and 80 μL of the present enzyme solution diluted appropriately was kept at 37° C. The diluted solution used was 20 mM HEPES-NaOH buffer (pH 7.5) containing 50 mM NaCl. After 60 minutes of reaction, 40 μL of 2.0 M HCl was added, and the reaction was stopped by keeping at 100° C. for 5 minutes. The operation after stopping the reaction was performed in the same manner as in Example 3-1, the amount of glucose generated by the reaction was determined, and the residual activity was measured. The activity before keeping at each pH was taken as 100%, and the remaining activity after keeping was expressed as a relative value (Figure 4B). This enzyme showed a remaining activity of 80% or more in the pH range of 4.6 to 8.4, demonstrating its stability.

3-3. Optimum temperature 200 μL of reaction solution consisting of 40 μL of 500 mM mannose, 80 μL of 100 mM Britton-Robinson buffer (pH 7.5 or 6.0), and 80 μL of the enzyme solution diluted appropriately was kept at 40 to 65 ° C. The diluted solution was 20 mM HEPES-NaOH buffer (pH 7.5) containing 50 mM NaCl or 20 mM sodium acetate buffer (pH 6.0) containing 50 mM NaCl. After 60 minutes of reaction, 40 μL of 2.0 M HCl was added and the reaction was stopped by keeping at 100 ° C for 5 minutes. The operation after stopping the reaction was performed in the same manner as in Example 3-1, and the amount of glucose generated by the reaction was determined. The enzyme showed maximum activity at reaction pH 7.5 and 50 ° C. The enzyme showed 80% or more of maximum activity in the range of 40 to 55 ° C. at reaction pH 7.5. Furthermore, the activity at reaction pH 6.0 tended to increase up to 60° C., and was lower up to 55° C. compared with the activity at reaction pH 7.5, but showed high values at 60° C. and 65° C. In Fig. 5A, the activity at reaction pH 7.5 and 50° C. was set to 100%, and the activity under each reaction condition was shown as a relative value.

3-4. Temperature stability 50 μL of the present enzyme solution, the pH of which was adjusted to 7.5 or 6.0, was kept at 30°C to 70°C for 60 minutes, and the remaining activity after the keeping was determined according to the following method. 200 μL of the reaction solution, consisting of 40 μL of 500 mM mannose, 80 μL of 200 mM HEPES-NaOH buffer (pH 7.5), and 80 μL of the present enzyme diluted appropriately, was kept at 37°C. 20 mM HEPES-NaOH (pH 7.5) containing 50 mM NaCl was used as the dilution solution. After 60 minutes of reaction, 40 μL of 2.0 M HCl was added, and the reaction was stopped by keeping at 100°C for 5 minutes. The operation after stopping the reaction was performed in the same manner as the method described in Example 3-1, and the amount of glucose generated by the reaction was determined and the remaining activity was measured. The activity before keeping at each temperature was set to 100%, and the remaining activity after keeping was expressed as a relative value (FIG. 5B). This enzyme was stable at pH 7.5, showing a residual activity of 90% or more at 50° C. or less, and at pH 6.0, showing a residual activity of 90% or more at 60° C. or less.

Example 4: Mannose synthesis test 1. Method Using the present enzyme solution, a mannose synthesis test using glucose as a substrate was carried out. That is, 1 mL of reaction solution consisting of 10.3 to 330.0 μL/g-ds of the present enzyme solution or an empty vector control, 50% glucose, 40 mM each buffer (MES-NaOH buffer (pH 6.0), sodium phosphate buffer (pH 7.0) or HEPES-NaOH buffer (pH 8.0)), and 0.02% sodium azide was kept at 50° C. for 24 to 72 hours. The reaction solution collected at each reaction time was adjusted to pH 4.0 or less and boiled for 10 minutes to stop the reaction. Then, 10 μL of the reaction solution was mixed with 800 μL of pure water to dilute it.

The sugar composition of the product of this enzyme using glucose as a substrate was analyzed by HPLC. The analysis conditions were as follows: column: HILICpak VG-50 4E (Shodex), HILICpak VG-50G 4A (Shodex), eluent: 80% acetonitrile, flow rate: 0.6 mL/min, column temperature: 40°C, detector: charged aerosol detector.

2. Results The results of sugar composition analysis of the reaction products obtained when the enzyme solution was allowed to act on glucose as described above are shown in Figures 6 and 7. The mannose content and fructose content in the reaction products were calculated from the peak area ratios in HPLC analysis.

In the sections where this enzyme solution was added, mannose was produced from glucose in all test sections at pH 6.0, 7.0, and 8.0. On the other hand, in the test section where the empty vector control was added, mannose was not produced at any of the pH values tested.

When 330.0 μL/g-ds of this enzyme solution was added and the reaction temperature was kept at 50°C for 72 hours, the mannose content was 27.0% at pH 6.0, 27.1% at pH 7.0, and 26.8% at pH 8.0.

In the pH 6.0 test group, fructose was barely detectable when either the enzyme solution or the empty vector control was used (0.7% or less when the enzyme solution was used). On the other hand, in the pH 7.0 and 8.0 test groups, fructose production was confirmed when either the enzyme solution or the empty vector control was used. When reacted for 72 hours at pH 7.0, the fructose content was 1.1-1.6% when either the enzyme solution or the empty vector control was used. When reacted for 72 hours at pH 8.0, the fructose content was 3.6-7.2% when either the enzyme solution or the empty vector control was used. At both pH 7.0 and pH 8.0, the fructose content in the reaction product tended to decrease as the amount of enzyme added increased.

When 330.0 μL/g-ds of this enzyme solution was added and allowed to act at pH 8.0 for 72 hours, the mannose content in the reaction product was 26.8%, the fructose content was 3.6%, and the fructose by-product in the reaction product was equivalent to approximately 13.4% of mannose. When 330.0 μL/g-ds of this enzyme solution was added and allowed to act at pH 7.0 for 72 hours, the mannose content in the reaction product was 27.1%, the fructose content was 1.4%, and the fructose by-product in the reaction product was equivalent to approximately 5.2% of mannose. On the other hand, when 330.0 μL/g-ds of this enzyme solution 1 was added and allowed to act at pH 6.0 for 72 hours, the mannose content in the reaction product was 27.0%, the fructose content was 0.7%, and the fructose by-product in the reaction product was equivalent to approximately 2.6% of mannose.

These results suggest that fructose is produced by a nonenzymatic reaction (e.g., isomerization of glucose to fructose due to the influence of a buffer or pH) or a reaction of a contaminating enzyme (conversion of glucose to fructose by an isomerase). Furthermore, it was revealed that the production of fructose can be suppressed by carrying out the reaction at a pH of 7.0 or less, especially at pH 6.0.

SEQ ID NO: 1: Amino acid sequence of a protein derived from Paldisphaera borealis SEQ ID NO: 2: Nucleotide sequence encoding a protein derived from Paldisphaera borealis SEQ ID NO: 3: Amino acid sequence of a protein derived from Paldisphaera borealis SEQ ID NO: 4: Nucleotide sequence encoding a protein derived from Paldisphaera borealis SEQ ID NOs: 5 to 8: Primer sequences

Claims (4)

An enzyme preparation for catalyzing an epimerization reaction of a sugar, comprising any one of the following proteins (a) to (c), wherein the sugar is any one selected from the group consisting of glucose, mannose, talose, galactose, and disaccharides having a β-1,4 bond.
(a) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 1 or 3;
(b) a protein having an amino acid sequence having 90% or more amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO: 1 or 3 and having an activity of catalyzing an epimerization reaction of sugar;
(c) A protein having an amino acid sequence in which one or several amino acids have been substituted, inserted, deleted and/or added in the amino acid sequence set forth in SEQ ID NO: 1 or 3, and having the activity of catalyzing an epimerization reaction of sugar. An enzyme preparation for catalyzing an epimerization reaction of a sugar, comprising a culture supernatant of a transformed cell into which any one of the following polynucleotides (d) to (f) has been introduced, wherein the sugar is any one selected from the group consisting of glucose, mannose, talose, galactose, and disaccharides having a β-1,4 bond, and having an activity of catalyzing an epimerization reaction of a sugar.
(d) a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4;
(e) a polynucleotide that hybridizes under stringent conditions with a complementary strand of a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4, and encodes a protein having an activity of catalyzing sugar epimerization;
(f) A polynucleotide having a base sequence identity of 90% or more to a polynucleotide consisting of the base sequence shown in SEQ ID NO: 2 or 4, and encoding a protein having the activity of catalyzing sugar epimerization. A method for producing a sugar epimerization reaction product, comprising the step of reacting a sugar substrate with the enzyme preparation according to claim 1 or 2 to obtain a sugar epimerization reaction product. A method for producing a food, feed, cosmetic, or pharmaceutical, comprising a step of obtaining a food, feed, cosmetic, or pharmaceutical using an epimerization reaction product of a sugar obtained by carrying out the method according to claim 3.