A novel ketose 3-epimerase

Abstract

The present invention provides a ketose 3-epimerase that is highly safe, active and heat-resistant and can be used in the food industry, a microorganism that produces it, and a method for producing ketose. The present invention provides a ketose 3-epimerase that can be obtained from a microorganism belonging to the genus Arthrobacter, which has a molecular weight of about 36 kDa as measured by SDS-PAGE and has the following substrate specificities (A) and (B). (A) It has the activity of diastereoisomerizing the 3-position of D- or L-ketose and generating the corresponding D- or L-ketose. (B) It has the highest diastereoisomerization activity for the 3-position of D-alulose in D- or L-hexulose.

Description

Technical Field

[0001] The present invention relates to a novel ketose 3-epimerase and a method for producing the same, a microorganism producing the same, a DNA encoding the enzyme and a recombinant vector and a transformed host cell containing the DNA, a ketose 3-epimerase mutant, and a method for producing ketose using the ketose 3-epimerase or the mutant. Background Art

[0002] Rare sugars are a general term for monosaccharides that do not exist in nature or exist only in trace amounts. Allose, altrose, gulose, idose, and talose, which are aldoses in hexose, are rare sugars, and allulose (psicose), sorbose, and tagatose, which are ketose, are rare sugars. In addition, if only the L-form is considered, L-fructose and L-glucose are also rare sugars.

[0003] Research on the production technology of rare sugars, as well as their physiological effects and chemical properties, is also ongoing. So far, their specific physiological effects have gradually been revealed, but in order to put them into practical use as medicines, pesticides, functional foods, etc., it is necessary to develop enzymes that react specifically with rare sugars and enable their mass production.

[0004] D-allulose, also known as D-psicose, is a diastereomer of D-fructose and has a sweetness of about 70% of that of granulated sugar. Its sweetness quality is similar to that of D-fructose. However, unlike D-fructose, D-allulose is known to be almost not metabolized when absorbed in the body, and its calories are close to zero. It has the function of inhibiting the activity of lipid synthase and thus reducing abdominal fat. So far, it has been reported that D-allulose can be used as a low-calorie sweetener (Patent Document 1) and a sweetener effective for weight loss (Non-Patent Documents 1 and 2). In Patent Document 2, focusing on the blood sugar rise inhibitory effect of D-allulose, it is recorded that it can be used in health foods, foods and beverages for diabetics, and foods and beverages for weight loss.

[0005] D-tagatose has a sweetness of about 90% of that of granulated sugar and an energy of 2 kcal / g, and is used as a low-calorie sweetener. It is also known to have an effect of increasing HDL cholesterol and inhibiting body fat accumulation (Patent Document 3), and has an effect of inhibiting postprandial blood sugar increase similar to D-alulose.

[0006] In the production of these rare sugars, methods using isomerases are currently used, which is the result of the discovery of useful enzyme reactions. Ketose 3-epimerase, one of the isomerases, can use a variety of ketoses as substrates, and sometimes the ketose with the highest diastereoisomerization activity at the 3rd position among the ketoses that will become substrates is included in its name, for example, the enzyme with the highest diastereoisomerization activity at the 3rd position of D-tagatose is called D-tagatose 3-epimerase. The technology of producing D-alulose, a rare sugar, from D-fructose by using this D-tagatose 3-epimerase (DTE) has been established.

[0007] For example, Non-Patent Document 3 discloses a Pseudomonas cichorii ) ST-24 strain D-ketose 3-epimerase, and it is described that D-alulose can be produced from D-fructose by using this enzyme. However, as indicated by its alias D-tagatose 3-epimerase, its activity for the diastereoisomerization of the 3-position of D-tagatose is the highest, and its activity for D-fructose is relatively weak.

[0008] In addition, Patent Document 4 discloses the use of Agrobacterium tumefaciens ( Agrobacterium tumefaciens Patent document 5 discloses a method for producing D-psicose (D-allulose) by using D-psicose 3-epimerase from Arthrobacter sphaeroides ( Arthrobacter globiformis ) have been proposed, however, none of these ketose 3-epimerases has high activity to the extent that D-allulose and D-tagatose can be mass-produced on an industrial scale.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent No. 4473980

[0012] Patent Document 2: Japanese Patent No. 5421512

[0013] Patent Document 3: Japanese Patent No. 5749880

[0014] Patent Document 4: Japanese Patent No. 4648975

[0015] Patent Document 5: Japanese Patent No. 5997693

[0016] Non-patent literature

[0017] Non-patent document 1: Asia Pac. J. Clin. Nutr. (2001) Vol. 10, p. 233-237

[0018] Non-patent document 2: Asia Pac. J. Clin. Nutr. (2004) Vol. 13, S127

[0019] Non-patent document 3: Biosci. Biotech. Biochem. (1993) Vol. 57, p. 1037-1039 Summary of the invention

[0020] Problems to be solved by the invention

[0021] The present invention aims to provide a novel ketose 3-epimerase, a method for producing the enzyme, a microorganism producing the enzyme, a DNA encoding the enzyme, a recombinant vector containing the DNA and a transformed host cell, an immobilized enzyme of the enzyme, a ketose 3-epimerase mutant protein with improved heat resistance, an immobilized protein of the mutant protein, and a method for producing ketose using the enzyme, the immobilized enzyme, the mutant protein and the immobilized protein, wherein the ketose 3-epimerase is derived from a microorganism that is approved for use in food production and is considered to be non-toxic, and has high activity and heat resistance, and can catalyze the isomerization from D-fructose to D-alulose with high activity.

[0022] Technical solutions to solve problems

[0023] The present inventors focused on bacterial species that are recognized for use as food not only in Japan but also in Europe and the United States and are listed as having little toxicity, and collected soil from various places, isolated microorganisms therefrom, and continued to search for microorganisms having ketose 3-epimerase activity.

[0024] As a result, a novel ketose 3-epimerase-producing strain belonging to Arthrobacter ( Arthrobacter ) genus. The Arthrobacter histidinolophilus microorganism ( Arthrobacter histidinolovorans ) The Y586-1 strain is able to produce a novel ketose 3-epimerase that is highly active and highly heat-resistant.

[0025] The novel ketose 3-epimerase from the microorganism has the activity of catalyzing the epimerization reaction at the 3-position of D- or L-ketose to produce the corresponding D- or L-ketose. The epimerization activity at the 3-position of D-alulose in D- or L-hexulose is the highest, and it is suitable for producing D-alulose from D-fructose.

[0026] That is, the present invention relates to the ketose 3-epimerase described in the following (1) to (5).

[0027] (1) a species from the genus Arthrobacter Arthrobacter) has a molecular weight of 36 kDa as measured by SDS-PAGE and has the following substrate specificities (A) and (B).

[0028] (A) It has the activity of diastereoisomerizing D- or L-ketose at the 3-position to generate the corresponding D- or L-ketose.

[0029] (B) Among D- or L-ketohexose, the epimerization activity toward the 3-position of D-alulose is the highest.

[0030] (2) The ketose 3-epimerase according to (1) above, which has the following physicochemical properties (C) and (D).

[0031] (C) The optimal reaction pH is 6-8.

[0032] (D) The optimal reaction temperature is 60℃.

[0033] The optimal reaction temperature is 60°C for the purified enzyme. In the examples, there are two experiments using the purified enzyme and the crude enzyme. The optimal reaction temperature of the crude enzyme is 70°C (refer to Figure 1 ). The crude enzyme is best at 70°C and the purified enzyme is best at 60°C, but it is also common that the crude enzyme has improved heat resistance due to the influence of surrounding proteins, etc. For recombinant mutants, for example, the description in paragraph 0047 of the International Publication that it is excellent in terms of being able to obtain multiple mutants with higher heat resistance than the wild-type ketose 3-epimerase can be understood as a crude enzyme.

[0034] (3) The ketose 3-epimerase according to (1) or (2) above, wherein the order of substrate specificity is D-allulose, D-tagatose, L-tagatose, D-fructose, and L-allulose.

[0035] (4) The ketose 3-epimerase according to any one of (1) to (3) above, wherein the microorganism belonging to the genus Arthrobacter is Arthrobacter histidinolophilus ( Arthrobacter histidinolovorans ).

[0036] (5) The ketose 3-epimerase according to any one of (1) to (4) above, wherein the microorganism belonging to the genus Arthrobacter is Arthrobacter histidinolophilus Y586-1 strain deposited with the International Patent Organisms Collection Center (Patent Microorganisms Collection Center) under the deposit number NITE BP-02813.

[0037] In addition, the present invention also relates to the proteins described in (6) to (8) below, the DNA, recombinant vector, or transformed host cell described in (9) to (13) below, or the microorganism described in (14) below.

[0038] (6) A protein, which is the protein described in the following (a) or (b).

[0039] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0040] (b) A protein consisting of an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 1, having the following ketose 3-epimerase activities (A) and (B), and wherein the ratio of the ketose 3-epimerase activities at reaction temperatures of 70° C. and 50° C. (T70 / T50) is 1.55 or more.

[0041] (A) It has the activity of diastereoisomerizing D- or L-ketose at the 3-position to generate the corresponding D- or L-ketose.

[0042] (B) Among D- or L-ketohexose, the epimerization activity toward the 3-position of D-alulose is the highest.

[0043] (7) A protein, which is the protein described in the following (a) or (b).

[0044] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO: 1.

[0045] (b) A protein consisting of an amino acid sequence having 70% or more identity with the amino acid sequence shown in SEQ ID NO: 1, having the following ketose 3-epimerase activities (A) and (B), and having a residual activity of 31% or more after incubation at 60° C. for 1 hour.

[0046] (A) It has the activity of diastereoisomerizing D- or L-ketose at the 3-position to generate the corresponding D- or L-ketose.

[0047] (B) Among D- or L-ketohexose, the epimerization activity toward the 3-position of D-alulose is the highest.

[0048] (8) A protein consisting of an amino acid substitution mutant of a protein consisting of the amino acid sequence shown in SEQ ID NO:1, wherein the protein has an amino acid substitution at at least one position selected from the group consisting of H56, T59, A60, P77, L94, L97, D101, A109, A128, V129, V132, S144, S167, P172, E199, A200, K202, and S218 in the amino acid sequence shown in SEQ ID NO:1, and has the following ketose 3-epimerase activities (A) and (B), and has a higher ratio (T70 / T50) of ketose 3-epimerase activities at reaction temperatures of 70°C and 50°C, or has a higher residual activity after incubation at 60°C for 1 hour, than the protein consisting of the amino acid sequence shown in SEQ ID NO:1.

[0049] (A) It has the activity of diastereoisomerizing D- or L-ketose at the 3-position to generate the corresponding D- or L-ketose.

[0050] (B) Among D- or L-ketohexose, the epimerization activity toward the 3-position of D-alulose is the highest.

[0051] (9) A DNA encoding the protein according to any one of (6) to (8) above.

[0052] (10) A DNA consisting of the base sequence shown in SEQ ID NO: 2 or a sequence complementary thereto.

[0053] (11) The DNA according to (9) above, which is the DNA according to any one of the following (a) to (c).

[0054] (a) a DNA consisting of a part or all of the base sequence shown in SEQ ID NO: 2 or a sequence complementary thereto,

[0055] (b) a DNA that hybridizes under stringent conditions with a complementary strand of a DNA consisting of the base sequence shown in SEQ ID NO: 2, and

[0056] (c) A DNA having a sequence identity of 70% or more to the DNA consisting of the base sequence shown in SEQ ID NO: 2.

[0057] (12) A recombinant vector comprising the DNA according to any one of (9) to (11) above.

[0058] (13) A transformed host cell obtained by transformation using the recombinant vector described in (12).

[0059] (14) An Arthrobacter histidinolophilus strain Y586-1 deposited with the International Patent Organism Collection Center (Patent Microorganism Collection Center) under the deposit number NITE BP-02813, which produces the ketose 3-epimerase described in any one of (1) to (3) above.

[0060] The present invention also relates to the immobilized enzymes described in the following (15) to (17), and the immobilized proteins described in the following (18) and (19).

[0061] (15) An immobilized enzyme comprising the ketose 3-epimerase according to any one of (1) to (5) above immobilized on a carrier.

[0062] (16) An immobilized enzyme comprising a crude ketose 3-epimerase immobilized on a carrier, wherein the crude ketose 3-epimerase is a crude ketose 3-epimerase present in a cell fragment of an Arthrobacter microorganism that produces the ketose 3-epimerase according to any one of (1) to (5).

[0063] (17) The immobilized enzyme according to (15) or (16) above, wherein

[0064] The above-mentioned carrier is an ion exchange resin, an organic polymer carrier and / or an inorganic carrier.

[0065] (18) An immobilized protein comprising the protein according to any one of (6) to (8) above immobilized on a carrier.

[0066] (19) The immobilized protein according to (18) above, wherein

[0067] The above-mentioned carrier is an ion exchange resin, an organic polymer carrier and / or an inorganic carrier.

[0068] The present invention also relates to methods for producing ketose 3-epimerase as described in (19) and (20) below, a method for producing a protein having ketose 3-epimerase activity as described in (21) below, and a method for producing ketose as described in (22) below.

[0069] (19) A method for producing ketose 3-epimerase, comprising culturing an Arthrobacter microorganism that produces the ketose 3-epimerase according to any one of (1) to (5) above in a culture medium, accumulating the ketose 3-epimerase in the microbial cells, and collecting the ketose 3-epimerase.

[0070] (20) The method for producing ketose 3-epimerase according to (19) above, wherein

[0071] The above-mentioned culture medium is an inorganic salt culture medium supplemented with D-allulose.

[0072] (21) A method for producing a protein having ketose 3-epimerase activity, comprising culturing the transformed host cell described in (13) above in a culture medium, and collecting the protein having ketose 3-epimerase activity from the culture.

[0073] (22) A method for producing ketose, wherein the ketose 3-isomerase described in any one of (1) to (5), the protein described in any one of (6) to (8), the immobilized enzyme described in any one of (15) to (17), or the immobilized protein described in (18) or (19) is allowed to act on a solution containing one or more selected from D- or L-ketose to cause diastereoisomerization at the 3-position of the ketose to generate the corresponding ketose and collect it.

[0074] Effects of the Invention

[0075] The ketose 3-epimerase of the present invention is characterized in that the activity is particularly high compared to existing ketose 3-epimerases derived from microorganisms. For example, compared to the ketose 3-epimerase derived from Arthrobacter sphaeroides described in Patent Document 5 ( Arthrobacter globiformis ) has an activity about twice as high per unit of culture solution as the ketose 3-epimerase produced by the present invention. Therefore, by using the enzyme obtained by the present invention, D-alulose can be efficiently produced in large quantities.

[0076] In addition, the optimal temperature of the enzyme is as high as 70°C, and even at 80°C, the relative activity is 59.4, about 60%, when the activity at the optimal temperature of 70°C is set to 100, which is highly heat-resistant. In addition, the residual activity is 49.4% even after heat treatment at 60°C for 1 hour, so a larger amount of product can be obtained in the reaction at the optimal temperature of 70°C. The excellent heat resistance of the enzyme can be further improved by preparing amino acid substitution mutants of the enzyme.

[0077] The advantage of using the enzyme of the present invention derived from a microorganism of the genus Arthrobacter in the food industry is the safety of the bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 This is a graph showing the optimal temperature of the crude enzyme of the present invention.

[0079] Figure 2 This is a graph showing the effect of culture medium on enzyme production.

[0080] Figure 3 The results of SDS-PAGE performed to confirm the molecular weight of the enzyme of the present invention are shown. In the figure, the unit of the numbers on the left is kDa.

[0081] Figure 4 This is a graph showing the optimal pH of the purified enzyme of the present invention and the control enzyme.

[0082] Figure 5 This is a graph showing the optimal temperature of the purified enzyme of the present invention.

[0083] Figure 6 This is a graph showing the temperature stability of the purified enzyme of the present invention and the control enzyme when incubated for 10 minutes.

[0084] Figure 7 This is a graph showing the substrate specificity of the purified enzyme of the present invention.

[0085] Figure 8 The relative activities of the immobilized enzymes of the present invention when HPA25L, WA30, IRA904CL, FRA54, FRA95, and XAD7HP are used as carriers are shown.

[0086] Fig. 9 To represent the 2+ The relative activities of the supports of each immobilized enzyme at 30°C to 80°C in the presence or absence of .

[0087] Fig.10 To express that Figure 3 The MALDI-TOF-MS analysis results of the fragments digested with trypsin after the 36 kDa band of SDS-PAGE was reduced and alkylated. That is, the peptide peak of the 36 kDa protein digested with trypsin obtained by MALDI-TOF-MS analysis.

[0088] Fig.11 The results of SDS-PAGE of the recombinant enzyme expressed in E. coli are shown in the figure. The recombinant enzyme is compared with the purified enzyme from Y586-1 strain by SDS-PAGE. The recombinant enzyme has an additional sequence of RSHHHHHH at the C-terminus and is about 1 kDa larger than the purified enzyme from Y586-1 strain. The units of the numbers on the left side of the figure are kDa.

[0089] Fig.12 This is a diagram showing the three-dimensional structure of the enzyme of the present invention (recombinant purified enzyme) obtained by X-ray crystal structure analysis. DETAILED DESCRIPTION

[0090] The present invention relates to a ketose 3-epimerase isolated from a microorganism belonging to the genus Arthrobacter, which has a high enzyme production per culture medium and has characteristic properties in terms of thermal stability.

[0091] Microorganisms of the genus Arthrobacter are safe microorganisms included in the Japanese food additive list as the origin of enzymes such as α-amylase, isomaltoglucanase, sucrase (invertase), urease, glucanase, α-glucosyltransferase, and fructosyltransferase, and as producers of trehalose and vitamin K (menadione).

[0092] Ketose is a chemical term used to classify sugars by their structure. It is a general term for monosaccharides containing a ketone group (keto carbonyl group) inside the chain structure. The molecular formula is C n H 2n O n (n≥3). A representative ketose is fructose, and the term also includes pentoses and the like.

[0093] In the present invention, ketose is a ketose including pentose and the like in a broad sense, and when described as ketohexose, it means a ketohexose of a hexose having a ketose structure. Specifically, it refers to fructose, allulose, tagatose, and sorbose, and D- or L-ketose refers to the D- and L-instances of these sugars.

[0094] Therefore, the ketose 3-epimerase of the present invention has the activity of isomerizing the 3-position of D- or L-ketose to generate the corresponding D- or L-ketose, and specifically, can catalyze the mutual conversion between D- or L-fructose and D- or L-alulose, and the mutual conversion between D- or L-tagatose and D- or L-sorbose. In addition, it can also act on all other ketoses.

[0095] The ketose 3-epimerase of the present invention can be prepared by the following method: Arthrobacter ) genus that has the ability to produce ketose 3-epimerase, and isolate ketose 3-epimerase from the bacteria grown in the culture solution. As a microorganism belonging to the genus Arthrobacter (Arthrobacter), for example, Arthrobacter histidinolophilus ( Arthrobacter histidinolovorans ) Y586-1 strain and its mutants, etc. At the time of this application, Y586-1 strain had been internationally deposited at the National Institute of Advanced Industrial Science and Technology International Patent Biological Depository (Institute for Product Evaluation and Technology Patent Microbiological Depository) located at 2-5-8, Kazusa Kamata, Kisarazu City, Chiba Prefecture, Japan on November 7, 2018, and was deposited under the deposit number NITE BP-02813.

[0096] Regarding strain Y586-1, we searched the 16S rRNA gene sequence of the strain using BLAST (Japan DNA database) and the known strains for homology. The result showed that the strain name with a homology of more than 98% was initially identified as Arthrobacter protocerae ( Arthrobacter protophormiae ). Later, the Japanese Food Analysis Center identified it as histidinolophilic Arthrobacter ( Arthrobacter histidinolovorans ), so a whole genome sequence search was performed on strain Y586-1, and the result confirmed that it was Arthrobacter histidinolophilus.

[0097] Therefore, a request for change of name of the strain with deposit number NITEBP-02813 was submitted to the International Patent Biological Depository of the National Institute of Advanced Industrial Science and Technology and was accepted on October 4, 2019.

[0098] The crude enzyme solution of the ketose 3-isomerase of the present invention can be prepared in the following manner: after aeration culture of the ketose 3-isomerase-producing bacteria of the genus Arthrobacter in an inorganic salt culture medium supplemented with D-alulose, the bacterial cells are recovered from the culture solution by centrifugation, the recovered bacterial cells are washed with 10mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride) buffer (pH7.5), and then suspended in 10ml of 10mM Tris-HCl buffer (pH7.5), lysozyme is added as lysozyme to break the cells by enzyme treatment, or the bacterial suspension is cooled in ice water while the cells are broken by an ultrasonic homogenizer, the broken products are centrifuged, and the centrifuged supernatant is taken.

[0099] The ketose 3-epimerase activity of the crude enzyme solution before purification can be confirmed by using D-fructose as a substrate and measuring the amount of D-allose produced.

[0100] The enzyme activity of the epimerization from D-alulose to D-fructose as the reverse reaction can also be measured under the same conditions. The conversion reaction of ketose is usually carried out under the following conditions. The substrate concentration is 1 to 60% (w / v), preferably about 5 to 50% (w / v), the reaction temperature is 10 to 70°C, preferably about 30 to 60°C, the reaction pH is 5 to 10, preferably about 7 to 10, and the reaction time can be appropriately selected. In the case of a batch reaction, it is usually selected from the range of 5 to 50 hours.

[0101] The crude enzyme solution is fractionated by changing the concentration of polyethylene glycol (PEG), and the supernatant containing PEG and ketose 3-epimerase is adsorbed to the target enzyme by ion exchange resin chromatography, and the PEG is washed away, and then the adsorbed enzyme is eluted. After that, the enzyme eluate is purified by ion exchange chromatography and hydrophobic chromatography in sequence, thereby separating and purifying the enzyme.

[0102] In order to confirm that the enzyme was purified, SDS-PAGE (gel concentration: 12.5%) was applied, and it was confirmed that a single band was obtained and the molecular weight was confirmed.

[0103] The ketose 3-epimerase of the present invention purified as described above has a molecular weight of about 36 kDa in SDS-PAGE, and is a metalloenzyme whose activation can be regulated by metal ions. The reaction with the substrate can be carried out in the presence of a metal ion selected from manganese, magnesium, iron, cobalt and aluminum at a concentration of 0.5 to 5 mM.

[0104] The ketose 3-epimerase of the present invention has a specified amino acid sequence. However, the gene of the enzyme of the present invention cannot be cloned using the PCR amplification method or the existing protein database, so the whole genome sequence of the Y586-1 strain was determined, and a database of protein groups encoding the whole ORF in the genome sequence was constructed. The fragments obtained by treating the purified enzyme with trypsin were analyzed using MALDI-TOF-MS, and because the amino acids of the obtained fragments were consistent, the amino acid sequence of the ketose 3-epimerase of the present invention was finally determined.

[0105] Examples thereof include a protein having the amino acid sequence shown in SEQ ID NO: 1, or a protein having an amino acid sequence homologous thereto and maintaining equivalent ketose 3-epimerase activity.

[0106] A homologous amino acid sequence refers to, for example, an amino acid sequence having 70% or more amino acid sequence identity with the amino acid sequence of SEQ ID NO: 1, preferably 75% or more, more preferably 76, 77, 78, 79 or 80% or more, even more preferably 85% or more, further preferably 86, 87, 88, 89, 90, 91, 92, 93 or 94% or more, and even more preferably 95%, 96%, 97%, 98% or more amino acid sequence identity.

[0107] The identity (%) of two amino acid sequences or two nucleic acid sequences (base sequences) can be determined, for example, by the following steps. First, the two sequences are placed side by side in a manner that allows for the most appropriate comparison. At this time, for example, a gap can be introduced into the first sequence to optimize the comparison with the second sequence. When the molecule (amino acid residue or nucleotide) at a certain position in the first sequence is the same as the molecule at the corresponding position in the second sequence, it can be said that the molecule at that position is identical. The identity of two sequences is a function of the number of identical positions in common in the two sequences (i.e., identity (%) = number of identical positions / total number of positions × 100), preferably also considering the number and size of the gaps required for optimization of the comparison.

[0108] In addition, comparison of two sequences and determination of identity should be achieved using a mathematical algorithm. As specific examples of mathematical algorithms that can be used for sequence comparison, the algorithm described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87: 2264-68 and improved in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-77 can be used, but it is not limited thereto. Such algorithms are incorporated into the NBLAST program and the XBLAST program (version 2.0) described in Altschul et al. (1990) J. Mol. Biol. 215: 403-10. In order to obtain a nucleotide sequence equivalent to the nucleic acid molecule of the present invention, for example, a BLAST nucleotide search can be performed using the NBLAST program with score = 100 and wordlength = 12.

[0109] The DNA of the present invention is a gene encoding the ketose 3-epimerase of the present invention, and has a predetermined base sequence. Examples thereof include a DNA sequence encoding the amino acid sequence shown in sequence number 1, a base sequence shown in sequence number 2, or a DNA sequence having a base sequence homologous to the base sequence shown in sequence number 2 and capable of encoding a protein that maintains the same ketose 3-epimerase activity as the protein of sequence number 1. A DNA consisting of the base sequence shown in sequence number 2 or a sequence complementary thereto is a novel compound.

[0110] The DNA involved in the present invention can be isolated by combining known techniques, such as combinatorial hybridization techniques or PCR techniques.

[0111] In addition, the DNA involved in the present invention also includes a DNA composed of the base sequence shown in SEQ ID NO: 2, a DNA that hybridizes with the DNA and its complementary chain under stringent conditions, and a DNA that has a sequence identity of 70% or more with the DNA and its complementary chain.

[0112] Here, stringent conditions refer to hybridization conditions that can be easily determined by a person skilled in the art. Generally speaking, they are experimental conditions empirically determined depending on the probe length, washing temperature and salt concentration. Usually, when the probe becomes longer, the temperature becomes higher for proper annealing, and when the probe becomes shorter, the temperature becomes lower. The hybridization formation between nucleic acids depends on the ability of complementary chains to reanneal in an environment slightly below their melting points. More specifically, as stringent conditions, as long as they can be selected by a person skilled in the art as conditions that can separate the above-mentioned DNA, there is no particular limitation. For example, it is preferred that in the washing stage, at a temperature of 42°C to 70°C, preferably at a temperature of 42°C to 65°C, the sodium concentration is 25mM to 450mM, preferably 25mM to 500mM. As such conditions, for example, conditions of washing at 42°C in 5×SSC (83mMNaCl, 83mM sodium citrate), 0.1% SDS, etc. can be cited. Moreover, by setting higher stringent conditions such as increasing the temperature, DNA with high homology can be obtained. Stringent conditions (stringent conditions) are to use a final 5×SSC, 0.2% SDS solution, and wash three times for 15 minutes each at 45°C (very low stringency), 50°C (low stringency), 55°C (intermediate high stringency), 60°C (moderately high stringency), 65°C (high stringency) and 70°C (very high stringency).

[0113] In addition, a homologous base sequence refers to "DNA having a sequence identity of 70% or more", and any percentage is acceptable as long as it is a DNA having a sequence identity of 70% or more with the base sequence of SEQ ID NO: 2. For example, it may refer to a base sequence identity of 70% or more, preferably 75% or more, more preferably 76, 77, 78, 79 or 80% or more, even more preferably 85% or more, further preferably 86, 87, 88, 89, 90, 91, 92, 93 or 94% or more, and even more preferably 95%, 96%, 97%, 98% or more.

[0114] It is also possible to insert the DNA of the present invention into an appropriate vector capable of autonomous replication to prepare a recombinant vector. The recombinant vector is composed of DNA and a vector capable of autonomous replication, and can be relatively easily prepared by recombinant DNA technology using conventional methods as long as the DNA can be obtained. An appropriate vector can be selected according to the purpose of use such as cloning or protein expression, or according to the host cell. Examples of such vectors include plasmid vectors such as pBR322, pUC18, pUB110, pTZ4, pC194, pHV14, TRp7, YEp7, and pBS7, and phage vectors such as λgt·λC, λgt·λB, ρ11, φ1, and φ105.

[0115] The recombinant vector obtained in this way can be introduced into appropriate host cells such as Escherichia coli, Bacillus subtilis, actinomycetes, yeast, etc. The introduction method uses a known method such as calcium phosphate co-precipitation, electroporation, lipofection, microinjection, etc. In order to obtain transformed host cells, colony hybridization method, etc. can be applied.

[0116] The method for producing the protein having ketose 3-epimerase activity of the present invention is not particularly limited, and a known method can be used. Specifically, the ketose 3-epimerase of the present invention can be produced by: culturing a microorganism having the ability to produce the ketose 3-epimerase of the present invention or a host cell transformed with a protein having ketose 3-epimerase activity of the present invention or a DNA encoding an amino acid substitution mutant of the protein described later in a nutrient medium, and collecting the protein having ketose 3-epimerase activity from the obtained culture. The culture method can use a known method, for example, both liquid culture and solid culture can be used.

[0117] After the bacterial cells are cultured as described above, the protein of the present invention is purified and recovered. The protein purification and recovery method can be freely selected from known methods. For example, when recovering from the culture solution, the protein can be separated and purified by, for example, removing insoluble matter from the culture supernatant by filtering, centrifuging, etc., and then appropriately combining various chromatography methods such as concentration using ultrafiltration membranes, salting out using ammonium sulfate precipitation, dialysis, ion exchange resins, etc. In addition, when recovering from the bacterial cells, for example, the bacterial cells are broken by lytic enzyme treatment, ultrasonic treatment, etc., and then separated and purified in the same manner.

[0118] In addition, the ketose 3-epimerase of the present invention can be used by immobilizing the enzyme, similar to other epimerases and isomerases, and an immobilized enzyme with high activity can be obtained by various immobilization methods. By using an immobilized enzyme, a large number of epimerization reactions can be carried out continuously, and the immobilized enzyme can be made by using known immobilization methods, such as carrier binding method, crosslinking method, gel encapsulation method, microencapsulation method, etc., and the carrier can be any carrier. In the carrier binding method, the enzyme is bound to a carrier (such as a derivative of a polysaccharide such as cellulose, dextran, agarose, polyacrylamide gel, polystyrene resin, porous glass, metal oxide, etc.) using physical adsorption, ionic bond and covalent bond. In the crosslinking method, the enzyme can be immobilized by crosslinking each other using a reagent having two or more functional groups. As cross-linking agents, glutaraldehyde for making Schiff bases, isocyanate derivatives for forming peptide bonds, N,N′-vinylmaleimide, bis(diazonium)benzidine for diazo coupling, or alkylated N,N′-polymethylenebis(iodoacetamide) can be used. In the gel encapsulation method, a lattice type in which the enzyme is contained in fine lattices of a polymer gel and a microcapsule type in which the enzyme is encapsulated by a polymer membrane of a semipermeable membrane can be used. In the lattice type method, polymer compounds such as synthetic polymers such as polyacrylamide gel, polyvinyl alcohol, and photocurable resins, and natural polymers such as starch, konjac flour, gelatin, alginic acid, and carrageenan can be used. In the microcapsule type method, hexamethylenediamine, sebacyl chloride, polystyrene, lecithin, etc. can be used. On the carrier, the above five enzymes can be fixed separately, or two, three, four, or five enzymes selected from these can be fixed in combination.

[0119] One embodiment of the method for immobilizing ketose 3-epimerase of the present invention is immobilization using a crude enzyme solution. Ketose 3-epimerase-containing crude enzyme solution obtained by ultrasonic treatment of a bacterial suspension is added to an ion exchange resin or the like and bound at low temperature to immobilize the ketose 3-epimerase.

[0120] In order to extract ketose 3-epimerase from bacteria, the bacterial cell wall needs to be disrupted, and there are disruption methods using ultrasonic treatment or enzyme treatment such as lysozyme as described above. It is known that a crude enzyme solution obtained by ultrasonic treatment contains a large amount of active ketose 3-epimerase.

[0121] As a carrier for immobilizing the enzyme from the crude enzyme solution obtained as described above, any carrier known as an immobilization carrier can be used. Various ion exchange resins, synthetic adsorbents, etc. are convenient and commonly used. In addition, a packaging method using sodium alginate can also be used.

[0122] That is, there is no particular limitation on the immobilization carrier used for the immobilization of ketose 3-epimerase as long as the enzyme can be immobilized. For example, organic polymer carriers such as ion exchange resins, polyvinyl alcohol, polypropylene, acrylic resins, chitosan, hydrophobic adsorption resins, chelate resins, and synthetic adsorption resins can be cited; inorganic carriers such as diatomaceous earth, diatomaceous earth, kaolinite, silica gel, molecular sieves, porous glass, activated carbon, and ceramics can be cited. In addition, when the microorganism is used as an immobilized enzyme, a pyridinium compound (for example, a trade name DAC manufactured by Denki Kagaku Kogyo Co., Ltd.) or alginate can be used as an immobilization carrier. These immobilization carriers can be used alone or in combination of two or more. Among these immobilization carriers, ion exchange resins are preferred from the viewpoints of maintaining the activity of ketose 3-epimerase by immobilization, low price, small pressure loss, and easy operation. For example, as a basic anion exchange resin, a strongly basic anion exchange resin or a weakly basic anion exchange resin can be used. For example, as a strongly basic anion exchange resin, HPA25L (manufactured by Mitsubishi Chemical Corporation) and IRA904CL (manufactured by ORGANO Corporation) can be listed, and as a weakly basic anion exchange resin, WA30 (manufactured by Mitsubishi Chemical Corporation), FPA54, FPA95 (manufactured by ORGANO Corporation) can be listed. As a synthetic adsorbent, XAD7HP (manufactured by ORGANO Corporation) can be listed.

[0123] When an immobilized enzyme is produced using a weakly basic anion exchange resin as a carrier, the immobilized ketose 3-epimerase is easily eluted after its isomerization energy disappears. Therefore, the immobilized carrier can be regenerated very easily, thereby reducing production costs.

[0124] As described above, in the same manner as the description of the immobilized enzyme in which the crude ketose 3-epimerase present in the bacterial cell fragment of the microorganism of the genus Arthrobacter that produces ketose 3-epimerase is immobilized on a carrier, in the present invention, as the immobilized ketose 3-epimerase, an enzyme in which the purified or roughly purified ketose 3-epimerase is immobilized can be used, and an enzyme in which the microorganism that produces ketose 3-epimerase is immobilized can also be used. Moreover, it is needless to say that, in the same manner as the crude enzyme and the purified enzyme, an immobilized protein in which an enzyme expressed by Escherichia coli is immobilized can be used.

[0125] The ketose 3-epimerase of the present invention is used to diastereoisomerize the 3-position of a ketose that can serve as a substrate, thereby converting it into a corresponding ketose, thereby producing the corresponding ketose. The ketose 3-epimerase of the present invention has a high diastereoisomerization activity for the 3-position of D-alulose or D-fructose, and thus can efficiently produce a large amount of D-alulose, a rare sugar, as a product from D-fructose that can serve as a substrate.

[0126] In addition, it is also possible to introduce mutations into the gene of the ketose 3-epimerase of the present invention, and to prepare mutants having various amino acid substitutions by replacing the corresponding amino acid residues with other amino acid residues through site-specific mutagenesis, thereby obtaining an enzyme having higher heat resistance than the wild-type enzyme without amino acid substitution. Heat resistance is evaluated by two indicators: the ratio of ketose 3-epimerase activity at reaction temperatures of 70°C and 50°C (T70 / T50) or the residual activity after incubation at 60°C for 1 hour.

[0127] According to the stereostructure analysis, the enzyme of the present invention is Fig.12 As shown, it is a homotetramer, and the active site residues of the enzyme of the present invention are predicted to be amino acids at positions 6, 63, 110, 152, 179, 182, 205, 211, and 240, and the metal ion binding site putative residues are predicted to be amino acids at positions 146, 177, 205, and 240. Therefore, amino acids near these amino acids are not substituted, but amino acids at other positions are substituted to prepare mutants.

[0128] Fig.12 This is a drawing showing the three-dimensional structure of the enzyme of the present invention (purified enzyme of the recombinant) obtained by X-ray crystallographic structural analysis. The purified enzyme of the recombinant was used for crystallization, and the structure of the ketose 3-dimerase from the Y586-1 strain was determined by X-ray crystallographic analysis. The enzyme of the present invention is a homotetramer (MolA, MolA, MolA, MolA) formed by the polymerization of 4 subunits. The structure of each subunit with a molecular weight of about 36 kDa confirmed by SDS-PAGE shows a β / α barrel structure, and a metal ion is bound to the active site in the center.

[0129] Surprisingly, even a mutant having an amino acid sequence identity of more than 70% with the ketose 3-isomerase of the present invention can maintain enzyme activity, and, compared with the wild-type enzyme of sequence number 1, the ratio of ketose 3-isomerase activity at reaction temperatures of 70°C and 50°C (T70 / T50) is higher, and the residual activity after incubation at 60°C for 1 hour is also higher.

[0130] In addition, the ketose 3-epimerase represented by SEQ ID NO:1 is similar to that of the previously reported Arthrobacter sphaeroides ( Arthrobacter globiformis ) derived from a ketose 3-dimerase (Patent Document 5), it was predicted that the position with different amino acid residues would contribute less to the activity of the ketose 3-dimerase, and the amino acids at the different positions in sequence number 1 were mutated by base substitution through site-specific mutagenesis, and various ketose 3-dimerase mutants were prepared, from which amino acid substitution mutants with higher heat resistance than the wild type were selected.

[0131] Site-specific mutation introduction methods can be performed using any method such as the inverse PCR method, the annealing method, etc. (Muramatsu et al., ed., "Revised 4th Edition New Genetics Engineering Handbook", Yodosha, p. 82-88). Various commercially available site-specific mutation introduction kits such as Stratagene's QuickChange II Site-Directed Mutagenesis Kit and QuickChange Multi Site-Directed Mutagenesis Kit can also be used as needed.

[0132] The most common method for site-specific mutation introduction is to use a mutation primer containing the nucleotide mutation to be introduced. Such a mutation primer can be designed as follows: annealing to the region containing the nucleotide sequence encoding the amino acid residue to be changed in the gene, and containing a base sequence that replaces the nucleotide sequence (codon) encoding the amino acid residue to be changed with a nucleotide sequence (codon) encoding the changed amino acid residue.

[0133] An amino acid substitution mutant refers to a mutant in which an amino acid in the original sequence is substituted with a different amino acid. There are conservative substitutions and non-conservative substitutions with respect to substitutions, and there is no particular limitation. In a preferred embodiment of the present invention, the substitution is a conservative substitution. Conservative substitutions are equivalent to substitutions between amino acids having the same properties (basic, acidic or neutral) and polarity (hydrophilic or hydrophobic), such as substitutions between aromatic amino acids or between aliphatic amino acids, such as substitutions between basic amino acids and basic amino acids, acidic amino acids and acidic amino acids, and polar amino acids and polar amino acids.

[0134] Conservative substitutions are made, for example, within the group of basic amino acids (Arg, Lys, His), acidic amino acids (Glu, Asp), neutral non-polar amino acids (Gly, Ala, Val, Leu, Ile, Met), aliphatic amino acids (Ala, Val, Leu, Ile, Met), polar amino acids (Gln, Asn, Ser, Thr), aromatic amino acids (Phe, Trp, Tyr), etc.

[0135] On the other hand, non-conservative substitution refers to the case of replacing an amino acid with a member outside the above group, for example, Cys is deleted or replaced with another amino acid in order to prevent folding in the protein in the three-dimensional structure. Alternatively, amino acid substitution is performed in order to maintain the balance of hydrophilicity / hydrophobicity or to increase the hydrophilicity in order to facilitate synthesis, taking into account the hydrophilicity index of amino acids (J. Mol. Biol. (1982) Vol. 157, p. 105-132) as an indicator of the hydrophobicity / hydrophilicity of amino acids.

[0136] In addition, there are cases where an amino acid having a smaller steric effect than the original amino acid is substituted, or a charged amino acid is substituted with an uncharged amino acid.

[0137] Substitutions include both conservative substitutions and non-conservative substitutions, for example, substitutions of Ala with Ser or Thr, substitutions of Arg with Gln, His or Lys, substitutions of Asn with Glu, Gln, Lys, His or Asp, substitutions of Asp with Asn, Glu or Gln, substitutions of Cys with Ser or Ala, substitutions of Gln with Asn, Glu, Lys, His, Asp or Arg, substitutions of Glu with Asn, Gln, Lys or Asp, substitutions of Gly with Pro, substitutions of His with Asn, Lys, Gln, Arg or Tyr, Ile is substituted with Leu, Met, Val or Phe, Leu is substituted with Ile, Met, Val or Phe, Lys is substituted with Asn, Glu, Gln, His or Arg, Met is substituted with Ile, Leu, Val or Phe, Phe is substituted with Trp, Tyr, Met, Ile or Leu, Ser is substituted with Thr or Ala, Thr is substituted with Ser or Ala, Trp is substituted with Phe or Tyr, Tyr is substituted with His, Phe or Trp, and Val is substituted with Met, Ile or Leu, but not limited to these. Regarding the substitution of these amino acid residues, the classification of the residues that can be substituted is only exemplified, and the amino acid residues that can be substituted are not limited by the classification.

[0138] Mutants with one amino acid substituted to 11 amino acids substituted and multiple mutants of the enzyme of the present invention were prepared and subjected to a heat resistance test. As a result, it was found that 30 out of 37 mutants had a higher ratio of ketose 3-dimerase activity at reaction temperatures of 70°C and 50°C (T70 / T50) than the mutant derived from Arthrobacter sphaeroides described in Patent Document 5 ( Arthrobacter globiformis)M30 control enzyme (1.54). In addition, regarding the residual activity after incubation at 60°C for 1 hour, 37 of the 37 mutants were higher than that from spherical bacteria (30.8%). Moreover, 36 of the 37 mutants were higher than the residual activity of the wild-type enzyme of the present invention represented by sequence number 1.

[0139] The ketose 3-epimerase of the present invention is also excellent in that a plurality of mutants having higher heat resistance than the wild-type ketose 3-epimerase can be obtained.

[0140] Example

[0141] Hereinafter, the present invention will be described in detail through experiments.

[0142] It should be noted that in the following experiments, there are cases where the D-allulose 3-epimerase activity that converts D-allulose to D-fructose is measured and recorded as ketose 3-epimerase activity, and cases where the D-fructose 3-epimerase activity that converts D-fructose to D-allulose is measured and recorded as ketose 3-epimerase activity. The present invention is not limited to the following examples in any way.

[0143] <Experiment 1: Origin and identification of strains>

[0144] The inventors of the present invention inoculated various bacteria isolated by screening into a liquid culture medium supplemented with D-allulose and cultured them with shaking, and measured the amount of D-allulose produced using D-fructose as a substrate, thereby measuring the activity of ketose 3-epimerase.

[0145] In this way, the microorganism Y586-1 strain was found as the strain with the highest activity, and it was determined to belong to Arthrobacter viridis through systematic analysis based on the homology of the 16S rRNA gene base sequence of the Y586-1 strain.

[0146] Identification of strains

[0147] (1) 16S rRNA gene base sequence homology

[0148] The 16S rRNA gene region was analyzed and the base number was determined to be 1,469 bp.

[0149] (2) Sameness search

[0150] The 16S rRNA gene base sequence of the strain was searched for homology with known species using BLAST search (Japan DNA database). Based on the strain name and homology (%) value with a homology of more than 98%, the microorganism Y586-1 strain was identified as Arthrobacter viridis ( Arthrobacter protophormiae), the Y586-1 strain was deposited internationally at the National Institute of Advanced Industrial Science and Technology International Patent Biological Depository on November 7, 2018, with the deposit number NITE BP-02813. Arthrobacter histidinolovorans ), therefore submitted an application for change of name of the strain with the deposit number NITE BP-02813 to the International Patent Biological Depository of the National Institute of Advanced Industrial Science and Technology, which was accepted on October 4, 2019.

[0151] <Experiment 2: Arthrobacter histidinolphaga ( Arthrobacter histidinolovorans ) Cultivation of strain Y586-1 (Deposit No. NITE BP-02813)

[0152] Arthrobacter histidinolophilus ( Arthrobacter histidinolovorans )Y586-1 strain (deposit number NITEBP-02813) seed culture solution 1% (v / v), and cultured at 30°C for 24 hours with ventilation and stirring. The ketose 3-epimerase activity in the obtained culture solution was about 2.694 units / 100 mL of culture solution. Hereinafter, this strain is abbreviated as "Y586-1 strain".

[0153] 1% (v / v) of the seed culture solution of the Y586-1 strain was added aseptically to each of the inorganic salt liquid medium, yeast extract liquid medium, broth liquid medium, and TSB liquid medium containing 1.0% D-allulose, and the culture was carried out at 30°C for 24 hours while aerating and stirring. The proliferation rate (OD 600nm ) and ketose 3-epimerase activity.

[0154] The results are shown in Figure 2 It was found that among the media studied, the enzyme production was high in the mineral salt medium where the proliferation was the lowest.

[0155] <Experiment 3: Preparation of crude enzyme>

[0156] The cells were recovered from the culture solution by centrifugation. The recovered cells were washed with 10 mM Tris-HCl buffer (pH 7.5). After that, the cells were suspended in 10 ml of 10 mM Tris-HCl buffer (pH 7.5), and the cell suspension was broken by using an ultrasonic homogenizer (Emerson Japan Co., Ltd.) while cooling in ice water. The broken product was centrifuged at 12,000 rpm for 20 minutes, and the supernatant was used as the crude enzyme.

[0157] <Experiment 4: Determination of crude enzyme activity>

[0158] The activity of ketose 3-epimerase of the enzyme before purification was determined by using D-alulose as a substrate and measuring the amount of D-fructose produced. Specifically, the enzyme reaction solution composition used 50mM Tris-HCl buffer (pH7.5), 0.1M D-alulose, enzyme solution, and 2mM magnesium chloride, and the reaction was carried out at 70°C for 10 minutes. The reaction was stopped by boiling for 2 minutes, and the composition of the solution after the reaction was determined by HPLC. One unit of enzyme activity is defined as the amount of enzyme that produces 1μmol of D-fructose in 1 minute by epimerizing D-alulose under the above conditions. The enzyme unit for epimerization from D-fructose to D-allulose, which is the reverse reaction, is also measured under the same conditions and defined in the same way.

[0159] <Experiment 5: Enzyme purification>

[0160] 1. Purification by ion exchange chromatography and hydrophobic chromatography

[0161] The crude enzyme solution was purified by ion chromatography. The chromatographic column used was HiTrapQ HP equilibrated with a buffer (20 mM Tris-HCl pH 7.5). The DUO FLOW system was used to extract 5 ml fractions at a flow rate of 5 ml / min with a concentration gradient of 1 M NaCl from 0% to 100%. The fractions with detected enzyme activity were recovered and separated by ion exchange chromatography to obtain purified enzyme.

[0162] 2. The enzyme purified by ion exchange chromatography was further purified by hydrophobic chromatography. The chromatographic column used was HiTrap PHENYL. Ammonium sulfate was added to the enzyme solution to make it 2M and dissolved. The concentration of 2M ammonium sulfate was reduced from 100% to 0% at a flow rate of 5 ml / min for elution. 5 mL was taken each time. The fractions in which the enzyme activity was detected were collected, and the ammonium sulfate was removed by dialysis to obtain the hydrophobic chromatography purified enzyme.

[0163] 3. Polyacrylamide gel electrophoresis

[0164] The purity of the purified enzyme was confirmed by SDS-PAGE (gel concentration 12.5%) according to conventional methods. Figure 3 In the figure, the left side is a protein with a standard molecular weight, and the middle column is an enzyme purified by hydrophobic chromatography. According to the results, a single band was confirmed at about 36 kDa, thereby confirming that the enzyme was purified purely. Regarding the molecular weight of the subunit of the purified enzyme of the present invention, the molecular weight obtained by SDS-PAGE was about 36 kDa.

[0165] <Experiment 6: Determination of physicochemical properties of crude and purified enzymes>

[0166] As a comparative enzyme, the enzyme derived from Arthrobacter sphaeroides described in Patent Document 5 was used. Arthrobacter globiformis ) Ketose 3-epimerase of M30. Hereinafter, it may be referred to as "control enzyme" and described as "AgDAE".

[0167] The enzyme reaction was carried out in the same experiment as in Experiment 4 for the determination of the activity of ketose 3-epimerase as crude enzyme and purified enzyme. The enzyme reaction was carried out for 10 minutes under each condition, and the amount of enzyme that produced 1 μmol of D-fructose from D-alulose in 1 minute was defined as 1 unit. After the reaction, the reaction solution was placed in a boiling liquid for 3 minutes to stop the reaction, and after deionization treatment, the produced D-fructose was determined by HPLC analysis.

[0168] 1. Optimal reaction temperature (wild strain crude enzyme)

[0169] The reaction was carried out at various temperatures ranging from 50 to 80°C. The reaction conditions for obtaining the optimal temperature are shown in Table 1. The temperature range from 60°C to 80°C is the preferred temperature for the crude enzyme. Figure 1 It can be seen that the optimal temperature of the crude enzyme of the enzyme of the present invention is when Mg is added. 2+ Ion is 70℃.

[0170] [Table 1]

[0171]

[0172] The optimum temperature of the crude enzyme of the control enzyme is also 70°C, but the crude enzyme of the enzyme of the present invention is Figure 1 As shown, particularly, with respect to the relative activity (%) when 70°C, which shows the highest activity, is set as 100, a relative activity of 77.6% is shown at 80°C, and a relative activity of 80.3% is shown at 60°C, while the crude enzyme of the control enzyme shows a relative activity of 58.7% at 80°C and a relative activity of 96.8% at 60°C. The enzyme of the present invention has a characteristic of being able to maintain its activity particularly at a temperature higher than the optimal temperature.

[0173] The optimal reaction temperature of the purified enzyme of the present invention (purified enzyme of wild strain) was obtained under the same reaction conditions. Figure 5 As shown, it is 60°C. In addition, Figure 6 The residual activity after the purified enzyme of the present invention and the control enzyme were incubated for 10 minutes. In the temperature stability after incubation for 10 minutes, the decrease in relative activity of the enzyme of the present invention at 60°C was smaller than that of the control enzyme, and the difference in the decrease in relative activity at 70°C was larger, which shows that the enzyme of the present invention is more stable at high temperatures than the control enzyme.

[0174] 2. Ketose 3-epimerase activity

[0175] The crude enzyme solution prepared in Experiment 3, 50 mM Tris-HCl buffer (pH 7.5), 0.1 M D-alulose, and 2 mM magnesium chloride were reacted at 70°C for 10 minutes. The reaction was stopped by boiling for 2 minutes, and the composition of the solution after the reaction was determined by HPLC. One unit (U) of enzyme activity is the amount of enzyme that diastereomerizes D-alulose under the above conditions and generates 1 μmol of D-fructose in 1 minute. The reaction conditions are shown in Table 2.

[0176] [Table 2]

[0177]

[0178] The results show that the total activity of the crude enzyme obtained from 100 mL of the culture solution was 2.694 U, while the total activity of the crude enzyme obtained from 100 mL of the culture solution was 0.847 U. The activity of the enzyme of the present invention was about 3 times higher than that of the control enzyme.

[0179] 3. Optimal pH of the reaction (purified enzyme from wild strain)

[0180] In the measurement, D-allulose was used as a substrate, and various buffer solutions at pH 4 to 11 were used to react at 70°C for 10 minutes. The generated D-fructose was measured by HPLC to determine the optimal pH for the reaction.

[0181] The reaction conditions are shown in Table 3. The buffer used is shown in Table 4.

[0182] [Table 3]

[0183]

[0184] [Table 4]

[0185]

[0186] The results are shown in Figure 4 The optimal reaction pH of the purified enzyme of the present invention is 6 to 8, whereas the optimal reaction pH of the control enzyme is 7.5 to 9, which is alkaline. The optimal reaction pH of the enzyme of the present invention is wider and exhibits high activity in more acidic conditions.

[0187] 4. Thermal stability (crude enzyme from wild strain)

[0188] The relative activity was determined by measuring the activity after heat treatment at 60°C for 1 hour, and the thermal stability of the enzyme was studied by setting the activity at 60°C as 100 under the reaction conditions (10 minutes) of the optimal temperature obtained in 2. shown in Table 2. As heat treatment conditions, a crude enzyme solution, a buffer solution (50 mM Tris-HCl pH 7.5), and 2 mM MgCl2 were used to prepare a total amount of 200 μl, which was kept at 60°C for 1 hour, cooled in ice water for 10 minutes, and the residual enzyme activity was determined according to a conventional method.

[0189] As shown in Table 7, the residual activity of the crude enzyme of the present invention after heat treatment at 60°C for 1 hour was 49.4%, whereas the residual activity of the control enzyme was 30.8%, indicating that the enzyme of the present invention has high thermal stability.

[0190] 5. Substrate specificity of ketose 3-epimerase (purified enzyme from wild strain)

[0191] The substrate specificity of the purified enzyme of the present invention was investigated using 8 kinds of ketohexose. The enzyme reaction set is shown in Table 5 below. The ketose produced by epimerization of each sugar was determined by HPLC analysis after reacting at 70°C for 10 minutes in a phosphate buffer solution with a final concentration of 50 mM and a final concentration of 0.1 M for each substrate.

[0192] The epimerization activity of D-alulose was set to 100, and the activities for each ketose were shown as relative activities.

[0193] D-fructose, D-allulose, D-sorbose, D-tagatose, L-fructose, L-allulose, L-sorbose, and L-tagatose were used as substrates, and the activities were expressed as relative activities, as shown in Tables 6 and 7. Figure 7 .

[0194] [Table 5]

[0195]

[0196] [Table 6]

[0197] Matrix Relative activity (%) D-Allulose 100.0 D-Fructose 43.4 D-Tagetang 72.2 D-sorbitol Trace L-Allulose 27.1 L-Fructose Trace L-Tagetang 58.2 L-sorbitol Trace

[0198] The enzyme of the present invention (purified enzyme of wild strain) has the strongest activity for D-allulose among the ketohexoses, followed by D-tagatose. When these substrate specificities are compared with other ketose 3-epimerases, they can also be called D-allulose 3-epimerase (DAE), showing a specificity significantly different from that of D-tagatose 3-epimerase (DTE) of Pseudomonas cichorii.

[0199] 6. Heat resistance (thermostability) test (recombinant crude enzyme)

[0200] (1) Ratio of ketose 3-epimerase activity at reaction temperatures of 70°C and 50°C (T70 / T50)

[0201] For the crude enzyme of the enzyme of the present invention (recombinant crude enzyme) and the control enzyme, the activity at 70°C and the activity at 50°C were measured under the reaction conditions (10 minutes) of the optimal temperature obtained in the above 2. shown in Table 2, and the ratio (T70 / T50) was obtained to explore the heat resistance.

[0202] The results are shown in Table 7. Regarding T70 / T50, the enzyme of the present invention (recombinant crude enzyme) was 1.78, and the control enzyme was 1.54, which showed that the enzyme of the present invention (recombinant crude enzyme) had relatively high activity at high temperatures.

[0203] (2) Residual activity after 1 hour at 60°C

[0204] For the crude enzyme of the enzyme of the present invention (recombinant crude enzyme) and the crude enzyme of the control enzyme, the relative activity was determined as an index of the heat resistance of the enzyme by measuring the activity after heat treatment at 60°C for 1 hour, when the activity at 60°C under the reaction conditions (10 minutes) of the optimal temperature obtained in the above 2. shown in Table 2 was set as 100. As the heat treatment conditions, the crude enzyme solution, buffer (50mM Tris-HCl pH7.5), and 2mM MgCl2 were made into a total of 200μl, which was kept at 60°C for 1 hour, cooled in ice water for 10 minutes, and then the residual enzyme activity was measured according to the conventional method.

[0205] The results are shown in Table 7. After incubation at 60°C for 1 hour, the residual activity of the enzyme of the present invention (recombinant crude enzyme) was 49.4, and the residual activity of the control enzyme was 30.8, indicating that the enzyme of the present invention (recombinant crude enzyme) has high thermal stability at 60°C.

[0206] [Table 7]

[0207]

[0208] <Experiment 7: Preparation of immobilized enzyme>

[0209] The following 1 to 5 are immobilization experiments using crude enzymes of wild-type strains, and bacterial cells of the Y586-1 strain cultured and recovered in the same manner as in Experiment 2 were used.

[0210] 1.Use ultrasonic treatment to obtain crude enzyme

[0211] 10 g (wet weight) of the bacterial cells were suspended in 10 ml of 50 mM Tris-HCl buffer (pH 7.5), and the cell suspension was disrupted by an ultrasonic homogenizer while cooling in ice water. The disrupted product was centrifuged at 12,000 rpm for 30 minutes, and the supernatant was used as a crude enzyme solution.

[0212] 2. Immobilization of crude enzyme

[0213] After being fully washed with pure water and swollen, the crude enzyme solution obtained above was added to an ion exchange resin or synthetic adsorbent equilibrated with 50 mM Tris-HCl buffer (pH 7.5) to allow the crude enzyme protein to bind thereto at 4°C for 24 hours. Subsequently, the immobilized enzyme was obtained by washing with 50 mM Tris-HCl buffer (pH 7.5).

[0214] As a preliminary experiment, strongly basic anion exchange resins (SA10A, SA11A, SA12A, NSA100, SA20A, PA306S, PA308, PA312, PA316, HPA25L, PA408, PA412, PA418 manufactured by Mitsubishi Chemical Corporation and IRA904CL manufactured by ORGANO Co., Ltd.), weakly basic anion exchange resins (WA10, WA20, WA21J, WA30 manufactured by Mitsubishi Chemical Corporation and IRA904CL manufactured by ORGANO Co., Ltd.) were used. The immobilization test was carried out on the ion exchange resin or synthetic adsorbent (FPA54, FPA60CL, FPA95) or synthetic adsorbent (XAD7HP, XAD118ON made by ORGANO). The activity expression rate of the immobilized enzyme obtained by adsorption of the crude enzyme solution on the ion exchange resin or synthetic adsorbent was more than 30%. The strong alkaline anion exchange resin HPA25L, IRA904CL, weak alkaline anion exchange resin WA30, FPA54, FPA95 and synthetic adsorbent XAD7HP were used for subsequent tests.

[0215] 3. Determination of enzyme activity of immobilized enzyme

[0216] The enzyme activity of the immobilized enzyme was measured by measuring the amount of D-fructose produced when an enzyme reaction was carried out using D-alulose as a substrate.

[0217] First, 100 mg of immobilized enzyme resin, Tris-HCl buffer (pH 7.5) (final concentration 50 mM) and D-alulose (final concentration 100 mM) solution (400 μl) were used as the reaction solution composition. After reacting in a 30°C constant temperature water bath for 10 to 30 minutes, the reaction was immediately stopped by boiling at 100°C for 2 minutes. The solution after the reaction was cooled to room temperature, desalted using an ion exchange resin (mixed resin of 200CT and IRA67 (both manufactured by ORGANO)), and further subjected to filter treatment to prepare an analysis sample. The analysis was performed by measuring the peak area of ​​the generated D-fructose using high performance liquid chromatography (chromatographic column: GL-C611 (Hitachi), temperature: 60°C, eluent: 0.1 mM NaOH, flow rate: 1.0 ml / min, detector: RID-20A (Shimadzu)).

[0218] 4. Comparison of enzyme activity caused by the carrier used for enzyme immobilization

[0219] In order to confirm the influence of the carrier for enzyme immobilization, the enzyme activity of the enzyme of the present invention immobilized on each carrier of HPA25L, WA30, IRA904CL, FPA54, FPA95, and XAD7HP was compared. It should be noted that the relative activity was calculated by taking the sample with the largest peak area among the comparison samples as 100.

[0220] Figure 8 The relative activity of various immobilized enzymes is shown. The activity of the immobilized enzyme produced using the synthetic adsorbent XAD7HP is the highest. As the amount of immobilized crude enzyme increases, the relative activity of each resin increases.

[0221] 5. Comparison of the enzymatic activity of immobilized enzymes induced by temperature

[0222] The reaction was carried out at various temperatures ranging from 30 to 80°C, and the relative activities of various immobilized enzyme carriers were determined. The results are shown in Fig. 9 .

[0223] Whether Mg is added 2+ The immobilized enzyme using XAD7HP as a synthetic adsorbent has relatively high activity at room temperature, while the immobilized enzyme using HPA25L, IRA904CL, and WA30 as an anion exchange resin has relatively high activity at high temperatures. It can be seen that the immobilization carrier should be selected according to the reaction temperature.

[0224] <Experiment 8: Cloning of DNA encoding enzyme and preparation of recombinant vector and transformed host cells containing the DNA>

[0225] From Arthrobacter histidinolophilus Arthrobacter histidinolovorans)Y586-1 strain cloned the DNA encoding ketose 3-epimerase, prepared a recombinant DNA capable of autonomous replication, determined the base sequence of the DNA encoding the enzyme, and prepared a transformed microorganism.

[0226] <Experiment 8-1: Determination of the complete base sequence of chromosomal DNA>

[0227] The ketose 3-epimerase of this bacterium is different from existing similar enzymes and cannot be isolated by PCR amplification methods or existing protein databases. Therefore, the whole genome sequence of the Y586-1 strain was determined, and a database of protein groups encoding the whole ORF in its genome sequence was constructed. The cultured cells of the Y586-1 strain were commissioned to Macrogen JAPAN Co., Ltd. for next-generation sequence analysis using PacBio-RSII / Sequel.

[0228] As a result, two contigs of 4,948,442 bp and 188,677 bp were obtained. These two contigs are about 5.1 Mb in size, so it is considered that they can cover the entire genome of Arthrobacter histidinolophilus ( Arthrobacter histidinolovorans )The complete genome sequence of the Y586-1 strain.

[0229] <Experiment 8-2: Construction of protein database>

[0230] Based on the obtained DNA sequence of about 5.1 Mb, 4,734 ORFs were estimated using the Prokka program, and the amino acid sequence was estimated for each ORF. These 4,734 amino acid sequences were used as a protein database for the Y586-1 strain.

[0231] <Experiment 8-3: Identification of proteins with ketose 3-epimerase activity>

[0232] Using the above protein database, we logged into the MASCOTserver (MatrixScience) protein identification system and identified proteins with ketose 3-epimerase activity. The 36 kDa band of SDS-PAGE in the international publication

[0049] was used as the test sample. After reduction and alkylation, the fragments digested with trypsin were subjected to MALDI-TOF-MS analysis ( Fig.10 ).

[0233] As a result, it was found that the protein had 87% similarity with the underlined amino acid sequence in the amino acid sequence of Sequence 1 (Sequence No. 1 in the sequence listing) composed of the following 289 amino acids, which strongly suggested that the protein of the present invention composed of the amino acids in Sequence No. 1 is the ketose 3-isomerase of the Y586-1 strain.

[0234] [Sequence 1] (Sequence number 1)

[0235] MKIGCHGLVWTGR FDAEGIRYSVQK TKEAGFDLIEFPLMDPFSFDVATAKSALAEHGLTASASLLGS EATDVSSEDPAIVKAGEELLNRALDVLAELGATDFCGVIYSAMKKYMEPATEQGLSNSK AAVGRVVDR AAGLGIN VSLEVVNRYETNVLNTGRQALSYLSDVKRPNLGVHLDTYHMNIEESDMFSPVLDTAEALKYVHIGESHRGYLGTGS VDFDNFFKALGRIGYDGPVVFESFSSAVVAPDLSRMLGIWRNLWADNEELGAHANAFIRDK LTAIKTIELH

[0236] (The amino acid sequence indicated by the underlined portion of the amino acid sequence identified using the protein database of the Y586-1 strain in Sequence 1 is a sequence consistent with the peak obtained by MALDI-TOF-MS.)

[0237] <Experiment 8-3: Isolation of ketose 3-epimerase gene>

[0238] The DNA sequence of the gene identified by the amino acid sequence (sequence number 2 in the sequence list) was synthesized and recombined into the pQE60 vector (Qiagen), and the expression was transformed into E. coli. The induction of the enzyme was confirmed using the constructed E. coli expression system. The results of SDS-PAGE are shown in Figure 2. Fig.11 As shown, an induced protein with RSHHHHHH added to the C-terminus was confirmed in the soluble fraction. The recombinant enzyme has an added amino acid sequence of RSHHHHHH at the C-terminus, so it is about 1 kDa larger than the purified enzyme of Y586-1 strain. In addition, the ketose 3-epimerase activity of this band was confirmed.

[0239] [Sequence 2] (Sequence number 2)

[0240] ATGAAAATTGGCTGCCACGGCCTGGTATGGACCGGCCGTTTCGACGCTGAAGGCATCCGCTACTCCGTCCAGAAGACCAAGGAAGCCGGGTTTGATCTGATCGAATTCCCGCTCATGGATCCCTTCTCCTTCGATGTGGCCACGGCAAAGTCGGCACTCGCAGAGCATGGTTTGACCGCCTCCGCATCATTGGGACTTTCCGAAGCTACGGATGTCAGCAGTGAAGATCCGGCCATTGTGAAAGCGGGGGAGGAGTTGCTCAACCGTGCCCTTGATGTTCTTGCGGAGCTGGGTGCCACCGACTTCTGCGGCGTGATCTACAGTGCGATGAAGAAGTACATGGAGCCGGCCACCGAGCAAGGCTTGAGCAACAGCAAGGCCGCTGTCGGGAGGGTGGTCGACAGGGCTGCTGGCCTGGGCATCAACGTCTCGCTTGAGGTCGTGAACCGGTATGAAACCAACGTGCTGAACACCGGGCGGCAGGCTCTGTCCTACCTCTCTGATGTCAAGCGGCCGAATCTAGGCGTTCATTTGGATACTTACCACATGAACATCGAGGAATCGGACATGTTCTCTCCGGTGCTGGACACCGCCGAGGCTCTTAAGTACGTGCACATTGGCGAGAGCCACCGAGGTTACCTCGGCACCGGGAGCGTCGATTTCGACAACTTCTTCAAGGCTCTTGGCCGCATCGGCTACGACGGACCGGTCGTCTTCGAATCATTCTCCTCCGCGGTCGTGGCCCCGGATCTGAGCCGCATGCTTGGCATCTGGCGCAACCTGTGGGCGGACAACGAAGAACTCGGCGCCCACGCCAACGCCTTCATACGCGACAAACTCACGGCCATCAAGACCATCGAACTGCAC TAA

[0241] (The DNA sequence shown by the underlined part identified using the protein database of strain Y586 - 1 for Sequence 2 had the bases relative to the stop codon removed during cloning.)

[0242] <Experiment 9: Mutants with an amino acid sequence identity of 70% or more to the enzyme of the present invention and their enzyme activities>

[0243] A mutant of the following sequence 3 (sequence number 3 in the sequence list) having an amino acid sequence identity of 78.5% with the amino acid sequence of sequence 1, and a mutant of the following sequence 4 (sequence number 4 in the sequence list) having an amino acid sequence identity of 76.8% were prepared, and the enzyme activity and heat resistance of the crude enzyme were measured using the same method as 2. and 6. of the above-mentioned experiment 6.

[0244] [SEQ ID NO. 3] (The underlined part is the amino acid different from SEQ ID NO. 1)

[0245] M N IGCHGLVWTG T FDA D GIR LA V E KTK Q AGFDLIEFPLMDPFSFDVA A A QA ALAEHGL AV SASLGLS DE TD IT S S DPA V V A AGE A LL L RA V DVLAELG G T H FCGVIYSAMKKYM D P V T PE GL AS S RRTIA RV A D H AA ER G VS VSLEVVNRYETNVLNT A RQAL A YL GE V D RPNLG I HLDTYHMNIEESDMF A PVLD A A P AL R YVHIGESHRGYLGTG T VDFD T FFKALGRIGYDGP I VFESFSSAVVAPDLSRMLGIWRNLWT DN D ELGAHANA Y IRDKL V A VDS I R LH

[0246] [SEQ ID NO. 4] (The underlined part indicates the amino acid different from that in SEQ ID NO. 1)

[0247] M N IGCHGLVWTG N FDAEGIR LA V H KT R EAGFDLIEFPLMDPF T FDV SV AK D AL E E YDIAV SASLGLSE E TDV T S A DPA VAA AGE A LL IK A V DVLA D LG GEH FCGVIYSAMKKYMEP V T PE GL AS SKAA I A RV A D H AA AR G VS VSLEVVNRYETNVLNT A RQAL A Y IAE V D RPNLG I H I DTYHMNIEESDMF T PVLD A A P AL R YVHIGESHRGYLGTG T VDFD T FFKALGRIGY N GP I VFESFSSAVVAPDLSRMLGIWRNLW N DN D ELGAHANA Y IRDKL V A VDS IR LH

[0248] The active site residues of the enzyme of the present invention are speculated to be amino acids at positions 6, 63, 110, 152, 179, 182, 205, 211, and 240, and the metal ion binding site residues are speculated to be amino acids at positions 146, 177, 205, and 240, so no amino acid substitution is performed near these amino acids.

[0249] The results of the recombinant crude enzyme are shown in Table 8 below.

[0250] [Table 8]

[0251]

[0252] In Table 8, Y586DAE1 refers to D-alulose 3-epimerase belonging to ketose 3-epimerase derived from strain Y586-1. The same shall apply hereinafter.

[0253] As shown in Table 8, it was confirmed that even for mutant enzymes having approximately 78% and 77% amino acid identity, as long as amino acid substitutions were made except in the vicinity of the active site and the metal ion binding presumed site, the enzyme activity could be maintained, and T70 / T50, which is an indicator of heat resistance, a useful property, would be increased, and the properties as an enzyme would not change.

[0254] <Experiment 10: Preparation of amino acid substitution mutants of the enzyme of the present invention and their enzyme activities>

[0255] The amino acid sequence of the ketose 3-epimerase represented by SEQ ID NO: 1 of the present invention was compared with that of a control enzyme (Arthrobacter sphaeroides ( Arthrobacter globiformis ) (Patent Document 5) was used to compare the amino acid sequence of ketose 3-dimerase (SEQ ID NO: 1) and it was inferred that the different amino acid residues at the different positions contribute less to the ketose 3-dimerase activity. The amino acids at the different positions of sequence number 1 were changed by base substitution through site-specific mutagenesis, and various ketose 3-dimerase mutants were prepared. From these, the positions that could produce amino acid substitution mutants with higher heat resistance than the wild type were selected.

[0256] First, a mutant with one amino acid substitution was prepared by site-specific mutagenesis. Two PCR primers with base sequences containing 1 to 2 base pairs of base substitutions at the target mutation introduction site were prepared, and a PCR reaction was performed with these two primers using plasmid DNA from the mutation introduction source. The obtained PCR fragment was transformed into the host Escherichia coli, plasmid DNA was extracted from the obtained clone, and the introduction of the target mutation was confirmed by sequence analysis. Multiple substitution mutants were prepared by repeating the above method.

[0257] The T70 / T50 and residual activity after 1 hour incubation at 60°C of the crude enzyme of each amino acid substitution mutant obtained by recombination were measured. Among them, mutants whose T70 / T50 or residual activity after 1 hour incubation at 60°C was higher than that of the control enzyme were selected, and amino acid substitutions at 18 positions in Table 9 were obtained.

[0258] [Table 9]

[0259] No Mutated amino acid number 1 H56 2 T59 3 A60 4 P77 5 L94 6 L97 7 D101 8 A109 9 A128 10 V129 11 V132 12 S144 13 S167 14 P172 15 E199 16 A200 17 K202 18 S218

[0260] Next, 2 to 11 mutants were prepared by simultaneously replacing 2 to 11 amino acids at the above 18 positions, and the T70 / T50 of the crude enzyme and the residual activity after incubation at 60°C for 1 hour of each recombinant mutant were measured. The results are shown in Tables 10 and 11.

[0261] Among the 37 species, 30 obtained site-specific mutant enzymes with T70 / T50 higher than the control enzyme's AgDAE (1.54), among which 20 obtained mutant enzymes with DAE higher than the parent wild-type recombinant enzyme Y586-1 strain (1.78) (Table 10).

[0262] In addition, 37 out of 37 species obtained site-specific mutant enzymes whose residual activity after incubation at 60°C for 1 hour was higher than that of the control enzyme AgDAE (30.8%), among which 36 out of 37 species obtained mutant enzymes whose residual activity was higher than that of the parent wild-type recombinant enzyme Y586-1 strain DAE (49.4%) (Table 11).

[0263] The above results show that the ketose 3-epimerase of the present invention is an excellent and useful enzyme that can obtain various mutants having improved thermal stability by performing site-specific mutation.

[0264] [Table 10]

[0265]

[0266] [Table 11]

[0267]

[0268] Industrial Applicability

[0269] The ketose 3-epimerase of the present invention can act on free D- or L-ketose, D- or L-ketopentose, and diastereoisomerize the 3-position of these ketoses to easily generate corresponding D- or L-ketose, D- or L-ketose. This reaction broadens the way for mass production of various ketoses, especially D-alulose using D-fructose as a raw material.

[0270] In particular, compared with the existing ketose 3-epimerase, the total activity obtained from the culture solution was about 3 times higher than that of the control enzyme. Therefore, the enzyme of the present invention can produce D-allulose in large quantities.

[0271] In addition, the enzyme has an optimum temperature of 70°C and is a highly heat-resistant enzyme, and thus can provide a higher production yield of D-allulose in a reaction at an optimum temperature of about 60 to 70°C.

[0272] In addition, the ketose 3-epimerase of the present invention can be immobilized to obtain an immobilized enzyme with high activity by various immobilization methods, and by using the immobilized enzyme, epimerization reactions can be continuously performed in large quantities. By enabling industrial immobilization, target ketose can be mass-produced.

[0273] Furthermore, the amino acid substitution mutants of the ketose 3-epimerase of the present invention include a plurality of enzymes having higher thermostability than the enzyme of the present invention before mutation.

[0274] Therefore, the establishment of the ketose 3-epimerase of the present invention and the method for producing the same is of great industrial significance not only to the sugar industry but also to the food, cosmetics, and pharmaceutical industries related thereto. Sequence Listing <110> National University Corporation Kagawa University <120> A novel ketose 3-epimerase <130> PCT19347UK <150> JP 2018-218287 <151> 2018-11-21 <160> 4 <170> PatentIn Version 3.5 <210> 1 <211> 289 <212> PRT <213> Arthrobacter histidinolovorans <400> 1 Met Lys Ile Gly Cys His Gly Leu Val Trp Thr Gly Arg Phe Asp Ala 1 5 10 15 Glu Gly Ile Arg Tyr Ser Val Gln Lys Thr Lys Glu Ala Gly Phe Asp 20 25 30 Leu Ile Glu Phe Pro Leu Met Asp Pro Phe Ser Phe Asp Val Ala Thr 35 40 45 Ala Lys Ser Ala Leu Ala Glu His Gly Leu Thr Ala Ser Ala Ser Leu 50 55 60 Gly Leu Ser Glu Ala Thr Asp Val Ser Ser Glu Asp Pro Ala Ile Val 65 70 75 80 Lys Ala Gly Glu Glu Leu Leu Asn Arg Ala Leu Asp Val Leu Ala Glu 85 90 95 Leu Gly Ala Thr Asp Phe Cys Gly Val Ile Tyr Ser Ala Met Lys Lys 100 105 110 Tyr Met Glu Pro Ala Thr Glu Gln Gly Leu Ser Asn Ser Lys Ala Ala 115 120 125 Val Gly Arg Val Val Asp Arg Ala Ala Gly Leu Gly Ile Asn Val Ser 130 135 140 Leu Glu Val Val Asn Arg Tyr Glu Thr Asn Val Leu Asn Thr Gly Arg 145 150 155 160 Gln Ala Leu Ser Tyr Leu Ser Asp Val Lys Arg Pro Asn Leu Gly Val 165 170 175 His Leu Asp Thr Tyr His Met Asn Ile Glu Glu Ser Asp Met Phe Ser 180 185 190 Pro Val Leu Asp Thr Ala Glu Ala Leu Lys Tyr Val His Ile Gly Glu 195 200 205 Ser His Arg Gly Tyr Leu Gly Thr Gly Ser Val Asp Phe Asp Asn Phe 210 215 220 Phe Lys Ala Leu Gly Arg Ile Gly Tyr Asp Gly Pro Val Val Phe Glu 225 230 235 240 Ser Phe Ser Ser Ala Val Val Ala Pro Asp Leu Ser Arg Met Leu Gly 245 250 255 Ile Trp Arg Asn Leu Trp Ala Asp Asn Glu Glu Leu Gly Ala His Ala 260 265 270 Asn With Phe Ile Arg Asp Lys Leu Thr Ala Ile Ile Lys Thr Ile Glu Leu 275 280 285 His <210> 2 <211> 870 <212> DNA <213> (Arthrobacter histidinolovorans) <400> 2 atgaaaattg gctgccacgg cctggtatgg accggccgtt tcgacgctga aggcatccgc 60 tactccgtcc agagaccaa ggaagccggg ttgatctga tcgaattccc gctcatggat 120 cccttctcct tcgatgtggc cacggcaaag tcggcactcg cagagcatgg tttgaccgcc 180 tccgcatcat tgggactttc cgaagctacg gatgtcagca gtgaagatcc ggccattgtg 240 aaagcggggg aggagttgct caaccgtgcc cttgatgttc ttgcggagct gggtgccacc 300 gacttctgcg gcgtgatcta cagtgcgatg aagaagtaca tggagccggc caccgagcaa 360 ggcttgagca acagcaaggc cgctgtcggg agggtggtcg acagggctgc tggcctgggc 420 atcaacgtct cgcttgaggt cgtgaaccgg tatgaaacca acgtgctgaa caccgggcgg 480 caggctctgt cctacctctc tgatgtcaag cggccgaatc taggcgttca tttggatact 540 taccacatga acatcgagga atcggacatg ttctctccgg tgctggacac cgccgaggct 600 cttaagtacg tgcacattgg cgagagccac cgaggttacc tcggcaccgg gagcgtcgat 660 ttcgacaact tcttcaaggc tcttggccgc atcggctacg acggaccggt cgtcttcgaa 720 tcattctcct ccgcggtcgt ggccccggat ctgagccgca tgcttggcat ctggcgcaac 780 ctgtgggcgg acaacgaaga actcggcgcc cacgccaacg ccttcatacg cgacaaactc 840 acggccatca agaccatcga actgcactaa 870 <210> 3 <211> 289 <212> PRT <213> Artificial Sequence <400> 3 Met Asn Ile Gly Cys His Gly Leu Val Trp Thr Gly Thr Phe Asp Ala 1 5 10 15 Asp Gly Ile Arg Leu Ala Val Glu Lys Thr Lys Gln Ala Gly Phe Asp 20 25 30 Leu Ile Glu Phe Pro Leu Met Asp Pro Phe Ser Phe Asp Val Ala Ala 35 40 45 Ala Gln Ala Ala Leu Ala Glu His Gly Leu Ala Val Ser Ala Ser Leu 50 55 60 Gly Leu Ser Asp Glu Thr Asp Ile Thr Ser Ser Asp Pro Ala Val Val 65 70 75 80 Ala Ala Gly Glu Ala Leu Leu Leu Arg Ala Val Asp Val Leu Ala Glu 85 90 95 Leu Gly Gly Thr His Phe Cys Gly Val Ile Tyr Ser Ala Met Lys Lys 100 105 110 Tyr Met Asp Pro Val Thr Pro Glu Gly Leu Ala Ser Ser Arg Arg Thr 115 120 125 Ile Ala Arg Val Ala Asp His Ala Ala Glu Arg Gly Val Ser Val Ser 130 135 140 Leu Glu Val Val Asn Arg Tyr Glu Thr Asn Val Leu Asn Thr Ala Arg 145 150 155 160 Gln Ala Leu Ala Tyr Leu Gly Glu Val Asp Arg Pro Asn Leu Gly Ile 165 170 175 His Leu Asp Thr Tyr His Met Asn Ile Glu Glu Ser Asp Met Phe Ala 180 185 190 Pro Val Leu Asp Ala Ala Pro Ala Leu Arg Tyr Val His Ile Gly Glu 195 200 205 Ser His Arg Gly Tyr Leu Gly Thr Gly Thr Val Asp Phe Asp Thr Phe 210 215 220 Phe Lys Ala Leu Gly Arg Ile Gly Tyr Asp Gly Pro Ile Val Phe Glu 225 230 235 240 Ser Phe Ser Ser Ala Val Val Ala Pro Asp Leu Ser Arg Met Leu Gly 245 250 255 Ile Trp Arg Asn Leu Trp Thr Asp Asn Asp Glu Leu Gly Ala His Ala 260 265 270 Asn Ala Tyr Ile Arg Asp Lys Leu Val Ala Val Asp Ser Ile Arg Leu 275 280 285 His <210> 4 <211> 289 <212> PRT <213> Artificial sequence <400> 4 Met Asn Ile Gly Cys His Gly Leu Val Trp Thr Gly Asn Phe Asp Ala 1 5 10 15 Glu Gly Ile Arg Leu Ala Val His Lys Thr Arg Glu Ala Gly Phe Asp 20 25 30 Leu Ile Glu Phe Pro Leu Met Asp Pro Phe Thr Phe Asp Val Ser Val 35 40 45 Ala Lys Asp Ala Leu Glu Glu Tyr Asp Ile Ala Val Ser Ala Ser Leu 50 55 60 Gly Leu Ser Glu Glu Thr Asp Val Thr Ser Ala Asp Pro Ala Val Ala 65 70 75 80 Ala Ala Gly Glu Ala Leu Leu Ile Lys Ala Val Asp Val Leu Ala Asp 85 90 95 Leu Gly Gly Glu His Phe Cys Gly Val Ile Tyr Ser Ala Met Lys Lys 100 105 110 Tyr Met Glu Pro Val Thr Pro Glu Gly Leu Ala Ser Ser Lys Ala Ala 115 120 125 Ile Ala Arg Val Ala Asp His Ala Ala Ala Arg Gly Val Ser Val Ser 130 135 140 Leu Glu Val Val Asn Arg Tyr Glu Thr Asn Val Leu Asn Thr Ala Arg 145 150 155 160 Gln Ala Leu Ala Tyr Ile Ala Glu Val Asp Arg Pro Asn Leu Gly Ile 165 170 175 His Ile Asp Thr Tyr His Met Asn Ile Glu Glu Ser Asp Met Phe Thr 180 185 190 Pro Val Leu Asp Ala Ala Pro Ala Leu Arg Tyr Val His Ile Gly Glu 195 200 205 Ser His Arg Gly Tyr Leu Gly Thr Gly Thr Val Asp Phe Asp Thr Phe 210 215 220 Phe Lys Ala Leu Gly Arg Ile Gly Tyr Asn Gly Pro Ile Val Phe Glu 225 230 235 240 Ser Phe Ser Ser Ala Val Val Ala Pro Asp Leu Ser Arg Met Leu Gly 245 250 255 Ile Trp Arg Asn Leu Trp Asn Asp Asn Asp Glu Leu Gly Ala His Ala 260 265 270 Asn Ala Tyr Ile Arg Asp Lys Leu Val Ala Val Asp Ser Ile Arg Leu 275 280 285 His

Claims

1. A ketose 3-epimerase derived from Arthrobacter histidinolovorans strain Y586-1 and consisting of the amino acid sequence shown in SEQ ID NO: 1, characterized in that: The molecular weight measured by SDS-PAGE is 36 kDa, and it has the following substrate specificity (A) and (B) and the following physicochemical properties (C) and (D). (A) having the activity of isomerizing the 3-position of D- or L-ketose to generate the corresponding D- or L-ketose, (B) The highest activity for the epimerization of D-alulose at the 3-position among D- or L-ketohexose is observed. (C) The optimal pH for the reaction is 6-8. (D) The optimal reaction temperature is 60℃.

2. An immobilized enzyme, characterized in that: The ketose 3-epimerase consisting of the amino acid sequence shown in SEQ ID NO: 1 according to claim 1 is immobilized on the carrier.

3. An immobilized enzyme, characterized in that: A crude ketose 3-epimerase is immobilized on a carrier. The crude ketose 3-epimerase is present in a bacterial cell fragment of an Arthrobacter microorganism that produces the ketose 3-epimerase having the amino acid sequence shown in SEQ ID NO: 1 according to claim 1.

4. The immobilized enzyme according to claim 2 or 3, characterized in that: The carrier is an ion exchange resin, an organic polymer carrier and / or an inorganic carrier.

5. A histidinolophagous Arthrobacter Y586-1 strain, characterized in that: It is deposited in the International Patent Biological Preservation Center with the deposit number NITE BP-02813, and produces the ketose 3-epimerase consisting of the amino acid sequence shown in the sequence number 1 as claimed in claim 1.

6. A protein, characterized in that: The present invention relates to a protein consisting of an amino acid substitution mutant of a protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and is a protein as described in the following (C), wherein the mutant has an amino acid substitution at at least one position selected from the group consisting of H56, T59, A60, P77, L94, L97, D101, A109, A128, V129, V132, S144, S167, P172, E199, A200, K202, and S218 in the amino acid sequence shown in SEQ ID NO: 1, and has the following (A) and (B) ketose 3-epimerase activity consisting of the amino acid sequence shown in SEQ ID NO: 1, and the ratio T70 / T50 of the ketose 3-epimerase activity at reaction temperatures of 70° C. and 50° C. is higher, or the residual activity after incubation at 60° C. for 1 hour is higher than that of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1, (A) having the activity of isomerizing the 3-position of D- or L-ketose to generate the corresponding D- or L-ketose, (B) The highest activity for the epimerization of D-alulose at the 3-position among D- or L-ketohexose is observed. (C)H56Y, L94V, L97M, D101E, A109S, A128V, V129I, V132I, S144A, S167Q, P172G, E199D, A200V, K202R, S218N, T59SA60VD101EV129IA200VS218 N. A200VS218N, D101EV129IA200V, T59SA60VA200VS218N, T59SA60VV129IA200V, T59SA60VA200V, V129IA200VS218N, T59SA60V, T59SA60VS21 8N, V129IS218N, V129IA200V, T59SA60VD101EA200V, T59SA60VD101EV129I, D101EA200V, T59SA60VV129IA200VS218N, T59SA60VV129I, T59SA 60VD101E, T59SA60VP77SL94VD101EA109SV129IS144AS167QA200VS218N, D101EV129I, D101EV129IA200VS218N, T59SA60VD101EA200VS218N.

7. An immobilized protein, characterized in that: The protein according to claim 6 is immobilized on a carrier.

8. The immobilized protein according to claim 7, characterized in that: The carrier is an ion exchange resin, an organic polymer carrier and / or an inorganic carrier.

9. A DNA encoding a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 or the protein according to claim 6.

10. A DNA, characterized in that: It consists of the base sequence shown in SEQ ID NO: 2 or a sequence complementary thereto.

11. A recombinant vector comprising the DNA according to claim 9.

12. A transformed host cell transformed by the recombinant vector according to claim 11.

13. A method for producing ketose 3-epimerase, characterized in that: An Arthrobacter microorganism producing the ketose 3-epimerase having the amino acid sequence shown in SEQ ID NO: 1 according to claim 1 is cultured in a medium, the ketose 3-epimerase is accumulated in the microbial cell, and the ketose 3-epimerase is collected.

14. The method for producing ketose 3-epimerase according to claim 13, wherein: The culture medium is an inorganic salt culture medium added with D-allulose.

15. A method for producing a protein having ketose 3-epimerase activity, characterized in that: The transformed host cell according to claim 12 is cultured in a culture medium, and a protein having ketose 3-epimerase activity is collected from the culture.

16. A method for producing ketose, characterized in that: In a solution containing one or more selected from D- or L-ketose, the ketose 3-epimerase composed of the amino acid sequence shown in SEQ ID NO. 1 according to claim 1, the protein according to claim 6, the immobilized enzyme according to claim 2 or 3, or the immobilized protein according to claim 7 is allowed to act to cause epimerization at the 3-position of the ketose to generate the corresponding ketose and collect it.

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