Method for producing ketoses using a novel ketose-3-epimerase

Abstract

The present invention aims to provide a method for producing allulose using an allulose-producing enzyme that exhibits high pH stability in the low pH range. [Solution] The present invention is a method for producing allulose, comprising the step of reacting fructose with a polypeptide consisting of a specific amino acid sequence or a polypeptide having 90% or more sequence identity with the above amino acid sequence and having allulose-producing ability, under acidic conditions of pH less than 6.

Description

Technical Field

[0006] , , ,

[0001] The present invention relates to a method for producing ketose using a novel ketose-3-epimerase having high pH stability in a low pH region.

Background Art

[0002] <000**********9>In recent years, due to the increasing health consciousness, the demand for rare sugars such as allulose, which are low in calories and have functionality, has been increasing.

[0003] Allulose can be synthesized from D-fructose by ketose-3-epimerase, including D-allulose-3-epimerase, D-tagatose-3-epimerase, and L-ribulose-3-epimerase, and allulose-producing enzymes derived from Pseudomonas cichorii and Arthrobacter globiformis are known (Patent Documents 1 and 2).

[0004] In the production of rare sugars such as allulose, reactivity in an acidic range is desired to prevent browning, but existing enzymes have low reactivity in the acidic range and are not suitable for treatment in the acidic range. Although it is known that an allulose-producing enzyme derived from Dorea sp. shows relatively high reactivity even in the acidic range, it is not sufficient (Non-Patent Document 1).

Prior Art Documents

Patent Documents

[0005]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0006]

Non-Patent Document 1

Summary of the Invention

[0007] The present invention aims to provide a method for producing ketoses using a novel ketose-3-epimerase that is acidophilic and acid-resistant. [Means for solving the problem]

[0008] The inventors diligently searched for an allulose-producing enzyme with high reactivity in acidic environments to prevent browning of allulose, a type of ketose and rare sugar. The inventors searched for microorganisms that produce allulose-producing enzymes with high reactivity in acidic environments from Amano Enzyme's internal strain library. As a result, they found a useful enzyme (conversion rate, reactivity in acidic environments) from Asaia krungthepensis, leading to the completion of the present invention.

[0009] In other words, the present invention is as follows. [1] A method for producing allulose, comprising the step of reacting fructose with a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6 or a polypeptide having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6 and having allulose-producing ability, under acidic conditions of pH less than 6. [2] The method of [1], wherein the acidic conditions below pH 6 are below pH 5. [3] The method of [1] or [2], wherein the temperature condition when reacting with fructose is 50°C to 70°C. [4] The polypeptide is derived from Asaia krungthepensis, by any of the methods [1] to [3]. [5] Any of the methods [1] to [4], wherein a microbial cell containing a gene encoding a polypeptide capable of producing allulose is brought into contact with fructose. [6] An enzyme preparation containing as an active ingredient a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6, or a polypeptide having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, and having allulose-producing ability. [7] An enzyme preparation comprising a microorganism containing a gene that encodes a polypeptide having an amino acid sequence represented by SEQ ID NO: 6 or a polypeptide having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6 and having allulose-producing ability. [8] An enzyme preparation of [6] or [7], wherein the polypeptide is derived from Asaia krungthepensis. This specification includes the disclosures of Japanese Patent Application No. 2020-171513, which forms the basis of the priority claim of this application. [Effects of the Invention]

[0010] The allulose-producing enzyme (AkDAE) derived from A. krungthepensis of the present invention is reactive even in acidic ranges below pH 5. It is also reactive in high temperature ranges above 50°C. When allulose is produced using AkDAE, browning can be prevented and allulose can be produced with a high conversion rate. [Brief explanation of the drawing]

[0011] [Figure 1] This figure shows the results of an allulose production test using A. krungthepensis lysate. [Figure 2] This figure shows the optimal pH for AkDAE. [Figure 3] This figure shows the pH stability of AkDAE. [Figure 4] This figure shows the optimal temperature for AkDAE. [Figure 5] This figure shows the thermal stability of AkDAE. [Figure 6] This figure shows the substrate specificity of AkDAE. [Figure 7] This figure shows the conversion rate of AkDAE to rare sugars. [Figure 8] It is a diagram showing the results of comparing the reactivity of AkDAE and a known allulose-producing enzyme under neutral pH and acidic pH conditions. [Figure 9] It is a diagram showing the results of an allulose production test using Escherichia coli cells.

Mode for Carrying Out the Invention

[0012] Hereinafter, the present invention will be described in detail. The present invention is a method for producing allulose using an allulose-producing enzyme (AkDAE) derived from A. krungthepensis.

[0013] Allulose is a monosaccharide having a structure represented by chemical formula (I) and is classified as a ketose. Allulose is also called psicose, and although it exists naturally, its amount is small, so it is called a rare sugar.

[0014]

Chemical formula

[0015] 1. Allulose-producing enzyme derived from A. krungthepensis The allulose-producing enzyme of the present invention is derived from Asaia krungthepensis belonging to the genus Asaia of the family Acetobacteraceae and can be isolated from A. krungthepensis. The allulose-producing enzyme derived from A. krungthepensis of the present invention is D-allulose-3-epimerase (abbreviation: AkDAE), which is a kind of ketose-3-epimerase.

[0016] The nucleotide sequence of the DNA encoding AkDAE is shown in SEQ ID NO: 5. Also, the amino acid sequence of the enzyme of the present invention is shown in SEQ ID NO: 6.

[0017] Insofar as the enzyme of the present invention has the activity to produce allulose using fructose as a substrate, at least one, preferably one or more, amino acids in the amino acid sequence may be subject to mutations such as deletion, substitution, or addition.

[0018] For example, at least one amino acid, preferably one or several (e.g., 1 to 10, more preferably 1 to 5, and especially preferably 1 or 2) may be deleted from the amino acid sequence represented by SEQ ID NO: 6, at least one amino acid, preferably one or several (e.g., 1 to 9, more preferably 1 to 5, and especially preferably 1 or 2) may be added to the amino acid sequence represented by SEQ ID NO: 6, or at least one amino acid, preferably one or several (e.g., 1 to 9, more preferably 1 to 5, and especially preferably 1 or 2) may be substituted with other amino acids.

[0019] Examples of such amino acid sequences include those in which one or several amino acids are deleted, substituted, or added to the amino acid sequence of SEQ ID NO: 6, which, when calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or similar tools (for example, default parameters), exhibit at least 85%, preferably 90%, more preferably 95%, more preferably 97%, more preferably 98%, and particularly preferably 99% or more sequence identity with the amino acid sequence of SEQ ID NO: 6.

[0020] Proteins having an amino acid sequence in which one or more amino acids are deleted, substituted, or added in the amino acid sequence of SEQ ID NO: 6 are substantially identical to proteins having the amino acid sequence of SEQ ID NO: 6.

[0021] Furthermore, the DNA encoding AkDAE in this invention also includes DNA that can hybridize with DNA consisting of a complementary sequence to the DNA consisting of the base sequence represented in Sequence ID No. 5 under the following stringent conditions, and which encodes a protein that has the activity to produce allulose using fructose as a substrate. Specifically, this refers to conditions under which the DNA can be identified by performing hybridization at 68°C in the presence of 0.7-1.0M NaCl using a DNA-immobilized filter, followed by washing at 68°C with a 0.1-2 times concentration SSC solution (1 times concentration SSC consists of 150 mM NaCl and 15 mM sodium citrate). Alternatively, this is DNA that can form a hybrid by transferring and immobilizing the DNA on a nitrocellulose membrane using Southern blotting, and then reacting it overnight at 42°C in a hybridization buffer [50% formamide, 4×SSC, 50 mM HEPES (pH 7.0), 10× Denhardt's solution, 100 μg / ml salmon sperm DNA].

[0022] Furthermore, DNA encoding a protein that has the activity to produce allulose using fructose as a substrate is also included in the DNA encoding AkDAE of the present invention, which has sequence identity of at least 85%, preferably 90%, more preferably 95%, more preferably 97%, more preferably 98%, and particularly preferably 99% or more when calculated using BLAST (Basic Local Alignment Search Tool at the National Center for Biological Information) or the like (for example, default, i.e., initial parameters) with the DNA consisting of the base sequence represented in Sequence ID No. 5.

[0023] The enzymatic properties of AkDAE are as follows: (1) Effect AkDAE produces allulose using fructose as a substrate. The conversion rate of fructose to allulose by AkDAE is 25% or more, preferably 27% or more, when using 30-60 (w / v)% fructose as a substrate.

[0024] (2) Optimal pH The optimal pH for AkDAE is 6-7, but it exhibits over 80% of its activity at pH 5-10. This optimal pH was measured using 50 mM acetate buffer (pH 4.0-6.0), 50 mM phosphate buffer (pH 6.0-7.0), 50 mM Tris-HCl buffer (pH 7.0-9.0), and 50 mM glycine-NaOH buffer (pH 9.0-11.0).

[0025] (3) pH stability AkDAE is stable in the pH range of 4 to 10, and even in the pH range of 3 to 10, more than 60% of its activity remains after treatment at 4°C for 24 hours. The remaining activity at pH 3.0 is 60% or more, preferably 65% ​​or more, compared to the activity at pH 7.0 (which is considered 100%). Furthermore, the remaining activity at pH 4.0 is 90% or more, preferably 95% or more, compared to the activity at pH 7.0 (which is considered 100%).

[0026] (4) Optimal temperature The optimal temperature for AkDAE is 55–65°C, but it exhibits over 80% of its activity at 45–75°C. The optimal temperature is measured using 50 mM Tris-HCl buffer at pH 7.0.

[0027] (5) Thermal stability AkDAE is a heat-resistant allulose-producing enzyme. When treated at high temperatures of 50°C or higher for 1 hour, its residual activity at 50°C and 60°C is approximately 100%, and its residual activity at 70°C is 65% or higher, preferably 70%.

[0028] (6) Substrate specificity AkDAE produces D-allulose when used as a substrate and D-sorbose when used as a substrate. Furthermore, the epimerase activity of AkDAE is reversible. Therefore, it produces D-fructose when used as a substrate and D-tagatose when used as a substrate. In terms of substrate specificity, reactivity to D-allulose was the highest, while reactivity to D-fructose was 60%.

[0029] (7) Rate of conversion to rare sugars The rare sugar conversion rate is the ratio of reactants to products when the enzymatic reaction reaches a plateau. For example, the rare sugar conversion rates for AkDAE are D-Frc:D-All = 70.9:29.1 and D-Tag:D-Sor = 69.4:30.6.

[0030] (8) Molecular weight The molecular weight of AkDAE was 32.2 kDa.

[0031] 2. Method for manufacturing AkDAE AkDAE can be produced by culturing A. krungthepensis, recovering it from the culture medium or cells, and purifying it.

[0032] The culture conditions and methods are not particularly limited as long as the enzyme is produced. That is, given that the enzyme is produced, methods and culture conditions suitable for culturing the microorganism used can be appropriately set. Either liquid culture or solid culture may be used as the culture method, but liquid culture is preferred. The culture conditions for liquid culture will be explained using this as an example.

[0033] The culture medium is not particularly limited as long as it is a medium on which the microorganisms used can grow. For example, a medium can be used that contains carbon sources such as glucose, sucrose, genthiobiose, soluble starch, glycerin, dextrin, molasses, and organic acids, as well as nitrogen sources such as ammonium sulfate, ammonium carbonate, ammonium phosphate, ammonium acetate, or peptone, yeast extract, corn steep liquor, casein hydrolysate, bran, and meat extract, and inorganic salts such as potassium salts, magnesium salts, sodium salts, phosphates, manganese salts, iron salts, and zinc salts. Vitamins and amino acids may be added to the medium to promote the growth of the transformants used. The pH of the medium should be adjusted to, for example, about 3 to 8, preferably about 4 to 7, and the culture temperature should be usually about 20 to 40°C, preferably about 25 to 35°C, and the culture should be carried out under aerobic conditions for 1 to 10 days, preferably about 3 to 6 days. As for the culture method, for example, the shaking culture method and the aerobic deep culture method using a jar fermenter can be used.

[0034] After culturing under the above conditions, the target enzyme is recovered from the culture medium or bacterial cells. When recovering from the culture medium, for example, insoluble matter can be removed by filtering or centrifugation of the culture supernatant, and then the enzyme can be obtained by separating and purifying it using various chromatography methods such as ultrafiltration, salting out by ammonium sulfate precipitation, dialysis, or ion exchange resins, in appropriate combinations. On the other hand, when recovering from bacterial cells, for example, the bacterial cells can be disrupted by pressurization or sonication, and then the target protein can be obtained by separating and purifying them in the same manner as above. Alternatively, the bacterial cells can be recovered from the culture medium beforehand by filtration or centrifugation, and then the above series of steps (disruption, separation, and purification of bacterial cells) may be performed.

[0035] Furthermore, recombinant AkDAE can be produced using recombinant DNA containing the AkDAE gene. For example, it can be prepared by transforming a suitable host cell (e.g., E. coli) with the DNA encoding this enzyme (specifically, DNA with the base sequence of Sequence ID No. 5), and recovering the protein expressed in the transformant. The recovered protein can then be prepared as appropriate according to the purpose. Obtaining this enzyme as a recombinant protein in this way allows for various modifications. For example, by inserting the DNA encoding this enzyme and other suitable DNA into the same vector, and then producing recombinant proteins using this vector, it is possible to obtain this enzyme consisting of recombinant proteins to which any peptide or protein is linked. Modifications such as the addition of sugar chains and / or lipids, or modifications that result in N-terminal or C-terminal processing, may also be performed. Such modifications make it possible to simplify the extraction and purification of recombinant proteins, or to add biological functions.

[0036] 3. Enzyme preparations The enzyme can also be provided in the form of an enzyme preparation. The enzyme preparation of the present invention contains the enzyme of the present invention as an active ingredient. Furthermore, the enzyme preparation of the present invention can be provided in the form of a microbial cell containing the enzyme of the present invention within its cells. The enzyme preparation contains a microbial cell containing a gene encoding the enzyme of the present invention within its cells. The microorganism is not particularly limited as long as it is a microorganism capable of expressing and accumulating the enzyme of the present invention within its cells. Examples include Escherichia coli, Bacillus bacteria, acetic acid bacteria, filamentous fungi, actinomycetes, and yeast. Escherichia coli is preferred. These microbial cells can be produced as transformants by introducing the enzyme gene into the microorganism. Transformants can be produced by known methods. The above transformants can be brought into contact with fructose and allowed to act on the fructose. The transformants express the enzyme within their cells, and the enzyme acts on the fructose. After the enzyme acts on the fructose and allulose is produced, the microorganism can be destroyed by ultrasound or the like, and the microbial fragments can be removed by centrifugation or the like. The enzyme preparation of the present invention may contain, in addition to the active ingredient (this enzyme), excipients, buffers, suspending agents, stabilizers, preservatives, antiseptics, physiological saline, etc. Excipients may include starch, dextrin, maltose, trehalose, lactose, D-glucose, sorbitol, D-mannitol, sucrose, glycerol, etc. Buffers may include phosphates, citrates, acetates, etc. Stabilizers may include propylene glycol, ascorbic acid, etc., or heavy metals such as manganese, cobalt, magnesium, or their salts. Preservatives may include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, etc. Antiseptics may include ethanol, benzalkonium chloride, para-hydroxybenzoic acid, chlorobutanol, etc. The content of the active ingredient (this enzyme) in this enzyme preparation is not particularly limited as long as the desired ketose can be produced. The enzyme preparation of the present invention is usually provided in solid form (for example, granules, powders, immobilized enzymes in which the enzyme is immobilized on a material capable of fixing the enzyme on or inside the surface of silica or porous polymers) or in liquid form.

[0037] 4. Uses of AkDAE AkDAE can be used to produce allulose using fructose as a substrate. Other ketoses can also be produced by changing the substrate. For example, D-sorbose can be produced using D-tagatose as a substrate, and D-tagatose can be produced using D-sorbose as a substrate. AkDAE has a low optimal pH and high heat resistance. Therefore, allulose can be produced under low pH and high temperature conditions. Reducing sugars containing allulose are prone to browning, and browning can be prevented in acidic conditions. Therefore, production at a low pH is preferable to prevent browning. Furthermore, under high temperature conditions, the solubility of the substrate increases, allowing for the use of higher substrate concentrations, and consequently, the production of high concentrations of allulose. The temperature conditions for the enzyme reaction are 30 to 80°C, preferably 40 to 70°C, more preferably 45 to 60°C, and the pH conditions are pH 4.0 or higher and less than pH 7.0, preferably pH 4.0 or higher and less than pH 6.0, even more preferably pH 4.0 or higher and less than pH 5.0, or pH 4.0 or higher and pH 4.9, pH 4.8, pH 4.7, pH 4.6 or lower.

[0038] When carrying out the enzymatic reaction, the substrate concentration is preferably 30-60 (w / v)%, and the reaction time is preferably 10-180 minutes, more preferably 10-120 minutes and 10-60 minutes.

[0039] When the reaction is carried out using 30-60 (w / v)% fructose as the substrate, the conversion rate to allulose is 20% or more, preferably 25% or more. [Examples]

[0040] The present invention will be specifically described by the following embodiments, but the present invention is not limited to these embodiments.

[0041] [Example 1] Allulose production test using crushed A. krungthepensis lysate D-allulose-producing bacteria were screened for and selected from a bacterial strain library. First, in the 1st screening, approximately 20,000 strains were selected down to 70 using TLC. The TLC conditions were as follows: the reaction solution was applied to 60 TLC silica gel plates (Merck-Millipore, Massachusetts, USA), and fructose and allulose were separated using -butanol:acetate:water (8:1:1) as the developing solvent. Methanol containing 0.5% (w / v)N-(1-naphthyl)ethylenediamine dihydrochloride and 5.0% (w / v)sulfinic acid was used as the chromogenic agent.

[0042] Next, the strain with the best allulose conversion rate was selected using HPLC. The HPLC conditions used were as follows: Mili-Q was used as the solvent, fructose and allulose were separated using MCI GEL CK08EC (300 × 8.0 mm I.D.; Mitsubishi Chemical Corporation, Tokyo, Japan) (flow rate 0.6 mL / min, temperature 75°C), and each isomerized sugar was detected using an evaporative light scattering detection system (Shimadzu, Kyoto, Japan). As a result, Asaia krungthepensis showed the best allulose conversion rate, with a conversion rate to allulose of 28.9% (Figure 1).

[0043] [Example 2] Sequence determination Degenerate primers (Fw 5'-ATGAACAAGCTYGGAYTSCAYGC-3' (SEQ ID NO: 1): Rv 5'-TCAGCGSTTRTGGCGYCCYAGCGC-3' (SEQ ID NO: 2)) were designed from homologous sequences of Asaia species. Using these primers and the A. krungthepensis genome, the putative allulose-producing enzyme gene (AkDAE gene) was amplified by PCR. Next, primers (Rv 5'-ATCCATATCTGCAGTCAAGCC-3' (SEQ ID NO: 3): Fw 5'-TCGATTTCCCGAGCATATTCC-3' (SEQ ID NO: 4)) were designed based on the amplified internal sequence, and the upstream and downstream sequences of the putative allulose-producing enzyme gene were determined, respectively. The amino acid sequence predicted based on the resulting AkDAE gene is shown below. MNKLGLHAFVWTAGWTPEDAAMAIRSTAELGYDLIEASTMDLKAFDVPATLRELEANRLGITMSFGLTADMDISSGDPERIRRGEAHLLDAISLARDVGATHVCGILYSAFQKYATPVTAEGVRGSIEVVRRVAEKAQASGITLGMEVVNRYESNVLNTARQAIAYVKRVEMPNVKLHLDCYHMNIEEADSAEAIREAGDLLGYFHTGDSHRGYLGSGSVDFPSIFRALESIQYQGPITFESFSSAVVGQPLEGILGIWRNLWEDSRDLAAHAKAFTDVQMKSAREALGRQKR (Sequence ID 6)

[0044] [Example 3] Conversion rate in high-concentration substrate solution (heterogeneous expression) The full-length AkDAE sequence was amplified by PCR and restricted enzyme digestion with NcoI. This was ligated into a pQE-60 vector, also restricted enzyme digested with NcoI, and transformed into E. coli JM109. AkDAE was induced in recombinant E. coli using 0.1 mM IPTG and purified with nickel-nitrilotriacetic acid agarose (QIAgen, Hilden, Germany). The allulose conversion rate of AkDAE to 30-60 (w / v)% fructose solution was investigated. The reaction was performed using 50 mM Tris-HCl buffer pH 7.5 and incubated at 50°C for 1 hour. The allulose conversion rates for 30-60 (w / v)% substrate solutions were 28.5%, 28.2%, 28.2%, and 28.4% (Table 1). These results reveal that AkDAE is the allulose-producing enzyme derived from A. krungthepensis, which was cloned in this study.

[0045] [Table 1]

[0046] [Example 4] Optimal pH (heterogeneous expression) The optimal pH for AkDAE was investigated. The buffer solutions used were as follows: 50 mM acetate buffer (pH 4.0-6.0), 50 mM phosphate buffer (pH 6.0-7.0), 50 mM Tris-HCl buffer (pH 7.0-9.0), and 50 mM glycine-NaOH buffer (pH 9.0-11.0) DAE activity was measured by assaying a 0.2 mL reaction solution containing 60.0 (w / v)% fructose and 50 μM purified AkDAE at 70°C for 1 hour. After the reaction, the solution was boiled at 100°C for 5 minutes. After cooling, fructose and allulose were detected by HPLC, and the AkDAE activity was determined by defining the amount of enzyme producing 1 mmol of allulose per minute as 1 unit. Relative activity was evaluated with the activity at pH 7.0 set to 100%. The results are shown in Figure 2. As shown in Figure 2, AkDAE showed a relative activity of 80% or more compared to the activity at the optimal pH at pH 5.0.

[0047] [Example 5] pH stability (heterogeneous expression) The pH stability of AkDAE was investigated. The buffer solutions used were as follows: 50 mM acetate buffer (pH 3.0-6.0), 50 mM phosphate buffer (pH 6.0-7.0), 50 mM Tris-HCl buffer (pH 7.0-9.0), and 50 mM glycine-NaOH buffer (pH 9.0-11.0) 50 μM purified AkDAE was dissolved in each pH buffer and treated at 4°C for 24 hours. After treatment, an assay was performed at 70°C for 1 hour using a 0.2 mL reaction solution containing 60.0 (w / v)% fructose and the pre-treated 50 μM purified AkDAE. After the reaction was complete, the samples were boiled at 100°C for 5 minutes. After cooling, fructose and allulose were detected by HPLC, and enzyme activity was determined by defining the amount of enzyme producing 1 mmol of allulose per minute as 1 unit. Relative activity was evaluated with the activity of the untreated sample set to 100%. The results are shown in Figure 3. As shown in Figure 3, AkDAE retained more than 90% activity in the pH range of 4 to 9, and more than 60% activity in the pH range of 3 to 10.

[0048] [Example 6] Optimal temperature The optimal temperature for AkDAE was investigated. The reaction temperature ranged from 30 to 80°C. DAE activity was assessed by assaying a 0.2 mL reaction solution containing 50 mM phosphate buffer pH 7.0, 60.0 (w / v)% fructose, and 50 μM purified AkDAE for 1 hour. After the reaction was complete, the solution was boiled at 100°C for 5 minutes. After cooling, fructose and allulose were detected by HPLC, and the AkDAE activity was determined by defining the amount of enzyme producing 1 mmol of allulose per minute as 1 unit. Relative activity was evaluated with the activity at 65°C set to 100%. The results are shown in Figure 4. As shown in Figure 4, AkDAE showed more than 80% activity in the temperature range of 45-70°C.

[0049] [Example 7] Thermal Stability The thermal stability of AkDAE was investigated. The buffer solution used was 50 mM Tris-HCl buffer with a pH of 7.5. The pretreatment temperatures were 50°C, 60°C, 70°C, and 80°C. The pretreatment times were 0, 15, 30, 45, and 60 minutes. After cooling the pretreated 50 μM purified AkDAE, it was assayed at 70°C for 1 hour in a 0.2 mL reaction solution containing 60.0 (w / v)% fructose and 50 μM purified AkDAE. After the reaction was complete, it was boiled at 100°C for 5 minutes. After cooling, fructose and allulose were detected by HPLC, and the activity of AkDAE was determined by defining the amount of enzyme producing 1 mmol of allulose per minute as 1 unit. The relative activity was evaluated with the activity of the untreated sample set to 100%. The results are shown in Figure 5. Even after treatment at 50°C or 60°C for 1 hour, the residual activity of AkDAE remained at 100%. At 70°C for 1 hour, the residual activity rate was 72%. These results indicate that AkDAE is a heat-stable allulose-producing enzyme.

[0050] [Example 8] Substrate specificity We investigated the substrate specificity of AkDAE. The substrates used were 60.0 (w / v)% D-allulose (D-All), D-fructose (D-Frc), D-tagatose (D-Tag), and D-sorbose (D-Sor). 50 mM Tris-HCl buffer, pH 7.5, was used as the buffer. A 0.2 mL reaction solution containing each substrate and 50 μM purified AkDAE was assayed at 70°C for 1 hour. After the reaction, the solution was boiled at 100°C for 5 minutes. After cooling, the substrates and products were detected by HPLC. The enzyme activity was determined by defining one unit as the amount of enzyme producing 1 mmol of isomerized sugar per minute from each substrate. Relative activity was evaluated, with the activity using D-allulose as the substrate set as 100%. The results are shown in Figure 6. AkDAE exhibited the highest substrate specificity for D-allulose. The relative activities for D-fructose, D-tagatose, and D-sorbose were 63.6%, 23.8%, and 11.5%, respectively.

[0051] [Example 9] Rare sugar conversion rate We investigated the conversion rate of AkDAE to rare sugars. The substrates used were 60.0 (w / v)% D-allulose (D-All), D-fructose (D-Frc), D-tagatose (D-Tag), and D-sorbose (D-Sor). A 50 mM Tris-HCl buffer with a pH of 7.5 was used as the buffer. A 0.2 mL reaction solution containing each substrate and 50 μM purified AkDAE was assayed at 70°C for 4 hours. After the reaction was complete, the solution was boiled at 100°C for 5 minutes. After cooling, the substrates and products were detected by HPLC, and the conversion rate was calculated from the substrate-to-product ratio. The results are shown in Figure 7. The rare sugar conversion rates by AkDAE were D-Frc:D-All = 70.9:29.1 and D-Tag:D-Sor = 69.4:30.6.

[0052] [Example 10] Comparison under neutral pH and acidic conditions The reactivity of AkDAE with known allulose-producing enzymes was investigated under neutral and acidic pH conditions. The enzymes used were 0.5 μM AkDAE (DAE from Asaia krungthepensis), 0.5 μM AgDAE (DAE from Arthrobacter globiformis), and 2.0 μM PcDTE (DAE from Pseudomonas cichorii). For neutral pH conditions, 50 mM Tris-HCl buffer pH 7.0 was used, and for acidic pH conditions, 50 mM acetate buffer pH 5.0 was used. The reaction conditions were 50°C for 0-60 minutes. The results are shown in Figure 8. Figure 8A shows the results under neutral pH conditions, and Figure 8B shows the results under acidic pH conditions. As a result, under pH 7.0 conditions, there was no difference in allulose production among the three enzymes, but under pH 5.0 conditions, only AkDAE produced allulose.

[0053] [Example 11] Allulose production test using E. coli cells Transformed Escherichia coli JM109 cells were prepared by introducing the allulose-producing enzyme gene shown in Sequence ID No. 1. Cells were collected from the culture medium of the transformants and washed twice with 50 mM Tris-HCl buffer (pH 7.5) to prepare wet cells. 50 mg of wet cells were added to 50 mM Tris-HCl buffer (pH 7.5) containing 60.0 (w / v)% D-fructose and mixed. The mixture was treated at 50°C for 60 minutes. Samples were taken every 10 minutes, cells were destroyed by sonication, and then the mixture was centrifuged at 40,000 rpm at 4°C for 10 minutes to remove cell debris. The samples after cell removal were analyzed by HPLC to measure the amount of allulose and fructose produced. The results are shown in Figure 9. The rare sugar conversion rate using purified AkDAE was approximately 29% (Figure 7), while the rare sugar conversion rate using E. coli cells for bioconversion was 34%.

[0054] [Example 12] Comparative test with related enzymes The allulose-producing ability of allulose-producing enzymes from acetic acid bacteria other than Asaia krungthepensis was compared with AkDAE. The amino acid sequence identity between the allulose-producing enzymes and AkDAE of each strain of acetic acid bacteria other than Asaia krungthepensis, as inferred from their genomic information, is as follows. Gluconobacter thailandicus (NBRC100600): Sequence identity 31.1% Acetobacter indonesiensis (NBRC16471): Sequence identity 26.8% Neoasaia chiangmaiensis (NBRC101099): Sequence identity 48.0% Swaminathania salitolerans (NBRC104436): Sequence identity 81.5% Each bacterial cell was inoculated into 20 mL of a culture medium prepared in a 100 mL Erlenmeyer flask (2(w / v)% allulose, 0.52(w / v)% ammonium sulfate, 0.24(w / v)% potassium dihydrogen phosphate, 0.56(w / v)% dipotassium hydrogen phosphate, 0.03(w / v)% magnesium sulfate, 0.1(w / v)% yeast extract (Difco)), and the culture was incubated with shaking at 30°C for 2 days. The culture medium was then sonicated to obtain a lysate. 0.8 mL of 10(w / v)% fructose solution (100 mM Tris-HCl (pH 7.5)) was mixed with 0.1 mL of water and 0.1 mL of the lysate, and the mixture was reacted at 50°C for 48 hours. The reaction was then stopped by treating the mixture at 100°C for 10 minutes. The amount of allulose in the reaction mixture was analyzed by HPLC.

[0055] [Table 2]

[0056] The results are shown in Table 2. AkDAE showed a higher allulose conversion rate compared to allulose-producing enzymes derived from other acetic acid bacteria.

[0057] [Example 13] Comparative test with related enzymes (comparison under acidic conditions) We compared the allulose-producing enzymes derived from acetic acid bacteria other than Asaia krungthepensis with AkDAE to determine their allulose-producing ability under acidic conditions. AkDAE-producing strains were compared with Neoasaia chiangmaiensis (NBRC101099) and Swaminathania salitolerans (NBRC104436). Each bacterial cell was inoculated into 20 mL of a culture medium prepared in a 100 mL Erlenmeyer flask (2(w / v)% allulose, 0.52(w / v)% ammonium sulfate, 0.24(w / v)% potassium dihydrogen phosphate, 0.56(w / v)% dipotassium hydrogen phosphate, 0.03(w / v)% magnesium sulfate, 0.1(w / v)% yeast extract (Difco)), and the culture was incubated with shaking at 30°C for 2 days. The culture medium was then disrupted using ultrasound to obtain a lysate. 10 μL of 5(w / v)% allulose solution (100 mM Tris-HCl (pH 7.5) or 100 mM sodium acetate (pH 5.0)) was mixed with 10 μL of water and 10 μL of the lysate, and the mixture was reacted at 60°C for 1 hour, after which the reaction was stopped by treating at 100°C for 10 minutes. The amount of fructose converted from allulose was quantified using the F-Kit D-glucose / D-fructose (Roche). The epimerase activity at pH 5.0 was compared to the activity at pH 7.5, which was set to 100.

[0058] [Table 3]

[0059] The results are shown in Table 3. Compared to allulose-producing enzymes from other acetic acid bacteria, AkDAE showed epimerase activity comparable to that under neutral conditions, even under acidic conditions. [Industrial applicability]

[0060] The novel ketose-3-epimerase AkDAE of the present invention can be used to produce ketoses, particularly allulose, a rare sugar. [Sequence Listing Free Text]

[0061] Sequence IDs 1-4: Primers All publications, patents, and patent applications cited herein shall be incorporated herein by direct reference.

Claims

[Claim 1] A method for producing allulose, comprising the step of reacting fructose with a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6, or a polypeptide having 90% or more sequence identity with the amino acid sequence represented by SEQ ID NO: 6, which has allulose-producing ability, under acidic conditions of pH less than 6.

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